Modulators of gtpases and their use

ABSTRACT

The present invention relates to molecules which function as modulators (i.e., inhibitors and agonists) of the Ras-homologous (Rho) family of small GTPases (e.g. Rac, Cdc42 and Rho GTPases) and their use to treat diseases, including cancers (including solid tumors-medulloblastoma, ovarian, breast, head and neck, testicular, prostate among others and hematologic malignancies-B cell lymphoma, where these GTPases are overexpressed or hyperactivated), sporadic and genetic diseases where activation of Rho GTPases plays a pivotal role (Menkes disease, rheumatoid arthritis, atherosclerosis, diabetes (type I), Huntington&#39;s disease and Alzheimer&#39;s disease) which are mediated through these proteins. Compounds according to the present invention may also be used as a therapy for the treatment of  Entamoeba  spp. or  Acanthamoeba  spp. infections, especially including  Entamoeba histolytica.

RELATED APPLICATIONS, CLAIM FOR PRIORITY AND GRANT SUPPORT

The present application claims the benefit of priority from provisionalapplication No. 61/397,864, filed Jun. 17, 2010, entitled “Ras-relatedGTPases as Targets of Non-Steroidal Anti-inflammatory Drugs, which isincorporated by reference in its entirety herein.

This invention was made with government support under grantsU54MH074425, U54MH084690, R03MH081231 and P30CA118100 awarded by theNational Institutes of Health. The government has certain rights in theinvention.

FIELD OF THE INVENTION

The present invention relates to molecules which function as modulators(i.e., inhibitors and agonists) of the Ras-homologous (Rho) family ofsmall GTPases (e.g. Rac, Cdc42 and Rho GTPases) and their use to treatdiseases, including cancers (including solid tumors-medulloblastoma,ovarian, breast, head and neck, testicular, prostate among others andhematologic malignancies-B cell lymphoma, where these. GTPases areoverexpressed or hyperactivated), sporadic and genetic diseases whereactivation of Rho GTPases plays a pivotal role (Menkes disease,rheumatoid arthritis, atherosclerosis, diabetes (type 1), Huntington'sdisease and Alzheimer's disease) which are mediated through theseproteins. Compounds according to the present invention may also be usedas a therapy for the treatment of Entamnoeba sp. infections, especiallyincluding Entamoeba histolytia, as well as other amoeba speciesresponsible for amoebic dysentery as well as other infections, e.g.,acanthamoebiasis of the eye caused by acanthamoeba spp.

BACKGROUND OF THE INVENTION

The Ras-homologous (Rho) family of small GTPases (Rac, Cdc42 and Rho)are key regulators of actin reorganization, cell motility, cell-cell andcell-extracellular matrix (ECM) adhesion as well as of cell cycleprogression, gene expression and apoptosis (FIG. 1) [1-8]. In many humancancers (including colon and breast), aberrant Rho-family signaling dueto changes in the GTPase itself or in its regulation loops is a criticalunderpinning of tumor growth and survival, invasion and metastasis[9-13] (FIG. 1). RhoA and RhoC correlate with advanced ovarian cancerand peritoneal dissemination [14; 15]. Although Rac1 and Cdc42 have beenrecognized as attractive therapeutic targets, specific Rac GTPaseinhibitors while effective in culture [16; 17] have not been translatedto clinical use and there are no established Cdc42 specific inhibitors.Lovastatin was shown to inhibit Rho GTPase and reduce ovarian metastasisin a xenograft model [15]. However, the use of statins to block GTPasemembrane association has met with only modest success due to their broadspectrum inhibition of protein prenylation resulting in pleiotropiceffects on many GTPases and pathways [18]. Furthermore, recent animalstudies wherein the effects of geranylgeranlytransferase type Ideficiency were analyzed revealed an unexpected hyperactivation of RhoGTPases and concomitant severe joint inflammation {Khan, 2011}. Thus,more specific agents for clinical application are urgently needed.

OBJECTS OF THE INVENTION

It is an object of the invention to provide compounds for modulating Rhofamily GTPases in patients or subjects.

It is another object of the invention to treat disease states and/orconditions which are medicated through targeting of Rho family GTPases.

It is yet another object of the invention to provide pharmaceuticalcompositions which may be used to modulate, especially inhibit GTPasesin patients or subjects.

It is still a further object of the invention to treat cancer,especially ovarian cancer and other cancers where GTPases areoverexpressed or hyperactivated utilizing compounds, compostions and/ormethods which are presented herein.

It is an additional object of the invention to provide methods fortreating sporadic and genetic diseases where activation of Rho GTPasesplays a pivotal role including in Makes disease, rheumatoid arthritis,atherosclerosis, diabetes (type 1), Huntington's disease and Alzheimer'sdisease.

It still another object of the invention to inhibit and/or an infectionof Acanthamoeba spp. or Entamoeba histolytica or a disease state orcondition where Acanthamoeba spp. Entamoeba histolytica is a causativeagent including amoebic dysentery; extraintestinal amnoebiasis, amoebicliver abscess; amoeba cutis acanthamoebiasis of the eye; and amoebiclung abscess.

Any one or more of these and/or other objects of the invention may bereadily gleaned from a description of the invention which follows.

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to compounds according to the chemicalstructure I

Wherein R¹ and R² are each independently H or a C₁-C₃ alkyl group;

R³ is a

group,

where Y is absent, O or S;

n is 0, 1, 2, 3, 4, 5 or 6;

R is H, a C₁-C₂₀ alkyl group or a phenyl group optionally substitutedwith a hydroxyl, halo or C₁-C₃ alkyl group;

R^(1a) and R^(1b) are each independently H or a CH₃ group with theproviso that at least one of R^(1a) and R^(1b) is a methyl group; and

said —(CH₂)_(n)— moiety is optionally substituted with a halo group (F,Cl, Br or I), a C₁-C₃ alkyl group or a hydroxyl group (preferably, themethylene group alpha to the carbon group containing the R^(1a) andR^(1b) substituents is substituted with a methyl or ethyl group);

R⁴ is H or a C₁ to C₄ alkyl group;

R⁵ is H, halo (F, Cl, Br or I), a C₁-C₆ linear or branch-chained alkylgroup (preferably a C₁-C₄ alkyl group), a C₁-C₆ alkoxy group or a

group where R₅ is an optionally substituted phenyl or a 5- or 6-memberedheteroaryl group, or R⁵, together with R⁶, forms an optionallysubstituted phenyl group (preferably a C₁-C₄ alkoxy, e.g. methoxy orethoxy substituted phenyl group) or an optionally substituted 5- or6-membered heteroaryl ring (preferably, an oxazole ring, preferablysubstituted with a ortho- or pan-halogen e.g. F, Cl, substituted phenylgroup) thus forming a bicyclic ring system; and

R⁶ is H, halo, C₁-C₆ linear or branch-chained alkyl group (preferably, aC₁-C₄ alkyl group), a C₁-C₆ alkoxy group or a

group where R₅ is an optionally substituted phenyl or a 5- or 6-memberedheteroaryl group, or R⁶, together with R⁵, forms an optionallysubstituted phenyl group or an optionally substituted 5- or 6-memberedheteroaryl ring, thus forming an optionally substituted bicyclic ringsystem, ora pharmaceutically acceptable salt, enantiomer, solvate or polymorphthereof.

In preferred aspects of the invention, R⁵ and R⁶ together form a phenylgroup which is optionally substituted by at least one methoxy group(preferably, furthermost from the R³ substituent as in naproxen,methallenestril and flunoxaprofen—see FIG. 10 hereof) and R⁴ is H. Inother aspects of the invention, R^(1a) is methyl and R^(1b) is hydrogenproviding a chiral center and the possibility of racemic mixtures andindividual enantiomers, each of which may be used in the presentinvention. In substituent R³, n is preferably 0 or 1 and Y is absent. Inalternative preferred embodiments, when R⁵ and R⁶ together form a phenylor heteroaryl group, in the R³ substitutent, Y is absent and n is 0 or 1and when n is 1, the methylene group is substituted with a C₁-C₃ alkyl(preferably ethyl) and R^(1a) and R^(1b) are both methyl. When n is 0,only one of R_(1a) and R_(1b) is H, thus forming a chiral center. Inalternative aspects of the invention where R⁵ and R⁶ do not form a ringto create a bicyclic group, Y in R³ is preferably 0 or absent, n is 0,1, 2, 3, or 4 (preferably 0 or 3), R^(1a) is methyl and R^(1b) is H(thus forming a chiral center). In these monocyclic embodiments, R⁵ is Hor a halogen group, preferably H, R⁶ is H or a C₁-C₆ alkyl group(preferably H or a C₃ or C₄ linear or branch-chained alkyl group, R⁴ ispreferably H and R¹ and R² are H or CH₃, preferably H.

The present invention also relates to specific compounds which may beused to modulate GTPases. These compounds include, for example,R-Naproxen, S-Naproxen, methallenestril, R-Flunoxaprofen,S-flunoxaprofen, R-Ibuprofen, S-Ibuprofen, S-Ketoprofen, R-Ketoprofen,gemfibrozil, ecabet, exetecan acid, R-Ketoralac, S-Ketoralac,tanomastat, mitiglinide, cicloxillic acid, fexofenadine, cilomilast,levocabastine, tiagabine, cinalukrast and mixtures thereof, includingpharmaceutically acceptable salts, enantiomers, racemic mixtures,solvates and polymorphs thereof. A number of these compounds or theirsalts is presented in attached FIG. 10.

The compounds which are disclosed herein find use to treat cancers,including solid and epithelial tumors exemplified in this disclosure byovarian cancer, among others, sporadic or genetic diseases related tohyperactivated membrane trafficking where Rho GTPases are important(e.g. Menkes disease, rheumatoid arthritis, atherosclerosis, diabetes(type I), Huntington's disease and Alzheimer's disease Huntington's), aswell as to inhibit entamoeba histolyica and/or to treat infectionscaused by entamoeba histolytica as well as other disease states orconditions of acanthamoeba spp., such as acanthamoebiasis of the eye.

Accordingly, the present invention relates to a method for modulating,including inhibiting a GTPase in a patient or subject in need ofmodulation wherein the GTPase is, in particular, a Rac (e.g. Rac1-3)GTPase or Cdc42, the method comprising administering to said patient orsubject an effective amount of a compound as set forth hereinabove. Themodulator is preferably an antagonist of GTPase. The present inventionalso relates to methods for modulating disease and/or conditions whichare mediated through GTPases, including treating and/or inhibiting theprogression of neurologic and inflammatory diseases dependent on RhoGTPases such as Menkes disease, rheumatoid arthritis, atherosclerosis,diabetes (type I), Huntington's disease and Alzheimer's disease; thegrowth of cancer, including inhibiting and/or reducing the likelihood ofthe metastasis of cancer comprising administering to a patient orsubject in need thereof an effective amount of at least one compound asotherwise disclosed herein. In the case of cancer, compounds accordingto the present invention may be coadministered with at least oneadditional anticancer agent to inhibit the growth of and/or otherwisetreat the cancer, including reducing the likelihood of metastasis of thetreated cancer.

Further embodiments relate to infectious disease, embodied as a methodfor inhibiting Entamoeba histolytica or Acanthamoeba spp. and/ortreating and/or reducing the likelihood of a disease state or conditionin which Entamoeba histolytica or Acanthamoeba spp. is an infectiveagent, said method comprising administering to a patient in need of aneffective amount of a GTPase inhibitor compound as described herein.Disease states and/or conditions in which Entamoeba histolytica is aninfective agent include, for example, amoebic dysentery; extraintestinalamoebiasis, amoebic liver abscess; amoeba cutis; and amoebic lungabscess. Disease states or conditions in which Acanthamoeba spp. is aninfective agent include acanthamoebiasis of the eye (amoeba cutisacanthamoebiasis of the eye), among others.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows Rac1 and Cdc42 GTPases integrate signaling pathways thatare important in cancer growth and metastasis. Activation of tyrosinekinase receptors, G-protein coupled receptors (GPCRs) and integrinscauses Rac and Cdc42 GTPases to bind GTP and membranes. The GTP-boundproteins interact with specific downstream effectors to promote actinreorganization that affect changes in cell motility, adhesion, cellgrowth, gene expression and apoptosis. GTPase functions in theregulation of proliferation, angiogenesis and metastasis are intimatelylinked to cancer development and progression. Adapted from [1].

FIG. 2 shows dysregulation of GTPase targets: Elevated Cdc42 and Rac1bin human ovarian tumor samples. (A-B) Ovarian tumor tissue microarrayswere purchased from US Biomax and stained for Cdc42 (pAb 10155-1-APProteinTech Group, Inc.), inset shows low magnification view with lessstaining of adjacent normal tissue. Tumor grade and type was confirmed,and immunoreactivity scoring was determined. (B) Statistical analysesshow p<0.001 for low vs. high grade tumors, Dr. E. Bedrick of the UNMCancer Center Biostatistics core. (C) Primary tumor cDNA samples(TissueScan Disease Tissue qPCR Arrays, OriGene) were screened for Rac1bexpression by qPCR. Normal vs. Gr. 2 p<0.05. (D) Rac1 and Cdc42 GTPaseactivities measured by GLISA in cells from fresh ovarian patient ascitesor after 48 h in culture.

FIG. 3 shows enantiomer selective inhibition of OPases. (A) GLISAmeasures cellular effects of small molecules on Rac1. Cells wereuntreated or incubated with the indicated compound (R-Nap=R-naproxen,enantiomers differ based on orientation of circled methyl group;6MNA-6-methoxy-2-naphthalene acetic acid) for the indicated times thentreated +/−10 ng/ml EGF for 2 min. Equal cell lysate protein was assayedfor activated Rac1 using a commercial, plate based Pak-binding assayaccording to manufacturer's instructions (GLISA, Cytoskeleton, Inc.).Similar results were obtained for Cdc42 and S-naproxen was inactive (notshown) (B) Docking predicts R-enantiomer-selective binding of naproxento GDP-bound Rac1. R-naproxen predicted to bind GDP-bound Rac1, but notGTP-bound conformation. S-naproxen is sterically blocked from binding(not shown). Rac1 crystal structure used to dock molecules via FRED(OpenEye).

FIG. 4 shows enantiomer selective inhibition of ovarian tumor cellmigration & aggregation. A) R-naproxen inhibits ovarian tumor cellmigration. OVCA 429 cell migration was measured under the indicatedconditions using modified Boyden chambers. *P<0.05 Similar results wereobtained for SKOV3ip and OVCA 433 cells. (B) R-ketorolac inhibitsovarian tumor cell migration. SKOV3ip cells were incubated for 24 hunder the indicated conditions and migration was measured as in (A).Comparable results were obtained for OVCA 429 cells. C) Inhibition ofaggregation. OVCA 429 cells were trypsinized and resuspended at 3×106cells/ml in medium containing drugs as indicated (100 μM). 25 μl dropswere suspended and imaged after 24 h. Comparable responses were observedin OVCA 433 and SKOV3ip cells.

FIG. 5 shows certain assays of GTPase and pathway inactivation by drugtreatment. (A) OVCA 433 cells were treated with 100 μM R-naproxen for 1h, fixed and stained for Rac1, actin and TiamI. (B) Quantification ofstaining shows a notable loss of Rac1 and Tiam membrane association withR-naproxen, but not S-naproxen or 6MNA treatment. (C) Total Rac proteinlevels were unchanged by treatment. (D) OVCA 429 cells plated onFITC-fibronectin extend invadopodia into the matrix and create holes dueto the activity of associated matrix metalloproteinases. (E) R-naproxen,but not 6MNA decreased invadopodia formation.

FIG. 6 shows that R-Naproxen reduces tumor number in xenograft model.Athymic nude mice were given an oral dose (10 mg/kg) of the indicatedcompounds in transgenic dough. Individual dosing was confirmed by directobservation. Mice were acclimated with placebo for 3 days and 1 dayprior to injections of human ovarian GFP-tagged SKOV3ip cells, mice wereleft on placebo or provided the indicated treatments. After 2 weeks oftumor growth the mice were sacrificed, necropsy was performed and imageswere taken of the peritoneal cavity. All tumors were counted by movinginternal organs with quantification given in the chart.

FIG. 7 shows biological processes involved in ovarian cancer metastasis.Rho family GTPases (Rac and Cdc42) contribute to metastaticdissemination and metastatic success as represented by survival of cellsin ascites, mesothelial anchoring and invasion; these events areparticularly relevant to residual and recurrent disease.MCA=multicellular aggregate.

FIG. 8, Table 1, shows four downstream effectors with keys roles intumor growth and metastasis. These downstream pathways would be expectedto be at least partially inactivated by GTPase inhibition and can beused to monitor compound efficacy.

FIG. 9, Table 2, evidences that the doses effective for GTPaseinhibition are doses that are within the dose ranges which areacceptable for human treatment based on COX inhibition). Table 2 showsserum concentrations and effective doses of drugs. Serum concentrations(maximum (C_(max)) and steady state (C_(ave))) are based on typical oraldosing (S-Naproxen 500 mg; R,S-ketorolac 30 mg; 6-MNA 1000 mg ofnabumetone) and derived from FDA product literature and primaryliterature (S-Naproxen, [40; 41]; ketorolac [23; 26; 39; 45; 46]; 6-MNA[43; 44; 47]. Note that an IV dose of 30 mg ketorolac achieves aC_(max), of 13.7 μM [46; 71]. IC₅₀ values for COX1/2 in human cells wereobtained from the literature (R and S-naproxen, [27; 72-74]; R andS-Ketorolac, [23; 26; 39; 46; 71; 75]; 6-MNA, [43; 44; 47], NSC23766,[89]. Migration IC₅₀ values were estimated from limited dose responsedata (FIG. 4) or calculated by GraphPad Prism5 (ketorolac). NA—notapplicable, no human dosing; ND=not detected, no COX inhibition; TBD=tobe determined.

FIG. 10 sets forth a number of conventional preferred bioactivecompounds which may be used in the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The following terms shall be used throughout the specification todescribe the present invention. Where a term is not specifically definedherein, that term shall be understood to be used in a manner consistentwith its use by those of ordinary skill in the art.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of those smaller ranges may independently be included in thesmaller ranges is also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either both ofthose included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “and” and “the” include plural references unless thecontext clearly dictates otherwise.

Furthermore, the following terms shall have the definitions set outbelow.

The term “patient” or “subject” is used throughout the specificationwithin context to describe an animal, generally a mammal, especiallyincluding a domesticated animal (i.e., not a laboratory test animal) andpreferably a human, to whom treatment, including prophylactic treatment(prophylaxis), with the compositions according to the present inventionis provided. For treatment of those infections, conditions or diseasestates which are specific for a specific animal such as a human patient,the term patient refers to that specific animal. In most instances, thepatient or subject of the present invention is a human patient of eitheror both genders.

The term “effective” is used herein, unless otherwise indicated, todescribe an amount of a compound or component which, when used withinthe context of its use, produces or effects an intended result, whetherthat result relates to the prophylaxis and/or therapy of an infectionand/or disease state or as otherwise described herein. The termeffective subsumes all other effective amount or effective concentrationterms (including the term “therapeutically effective”) which areotherwise described or used in the present application.

The term “compound” is used herein to describe any specific compound orbioactive agent disclosed herein, including any and all stereoisomers,individual optical isomers or racemic mixtures, pharmaceuticallyacceptable salts and prodrug forms. Within its use in context, the termcompound may refer to a single compound or a mixture of compounds asotherwise described herein.

The term “bioactive agent” refers to any biologically active compound ordrug which may be formulated for use in the present invention. Exemplarybioactive agents include the compounds according to the presentinvention which are used to modulate GTPases and to treat cancer, otherdisease states and/or conditions as well as infections caused byEntamoeba sp. or Acanthamoeba spp. as well as other compounds or agentwhich are otherwise described herein.

The terms “treat”, “treating”, and “treatment”, are used synonyumouslyto refer to any action providing a benefit to a patient at risk for orafflicted with a disease, including improvement in the condition throughlessening or suppression of at least one symptom, delay in progressionof the disease, prevention or delay in the onset of the disease, etc.

Treatment, as used herein, encompasses both prophylactic and therapeutictreatment. Compounds according to the present invention can, forexample, be administered prophylactically to a mammal in advance of theoccurrence of disease to reduce the likelihood of that disease.Prophylactic administration is effective to reduce or decrease thelikelihood of the subsequent occurrence of disease in the mammal, ordecrease the severity of disease that subsequently occurs.Alternatively, compounds according to the present invention can, forexample, be administered therapeutically to a mammal that is alreadyafflicted by disease. In one embodiment of therapeutic administration,administration of the present compounds is effective to eliminate thedisease and produce a remission or substantially eliminate an Entamoebaor Acanthamoeba infection as otherwise described herein; in anotherembodiment, administration of the compounds according to the presentinvention is effective to decrease the severity of the disease orlengthen the lifespan of the mammal so afflicted, in the case of cancer,as well as sporadic and genetic diseases that are Rho GTPase driven,including for example, Menkes disease, rheumatoid arthritis,atherosclerosis, diabetes, Huntington's disease and Alzheimer's disease,among others.

The term “pharmaceutically acceptable” as used herein means that thecompound or composition is suitable for administration to a subject toachieve the treatments described herein, without unduly deleterious sideeffects in light of the severity of the disease and necessity of thetreatment.

The term “inhibit” as used herein refers to the partial or completeelimination of a potential effect, while inhibitors are compounds thathave the ability to inhibit.

The term “prevention” when used in context shall mean “reducing thelikelihood” or preventing a condition or disease state from occurring asa consequence of administration or concurrent administration of one ormore compounds or compositions according to the present invention, aloneor in combination with another agent. It is noted that prophylaxis willrarely be 100% effective; consequently the terms prevention and reducingthe likelihood are used to denote the fact that within a givenpopulation of patients of subjects, administration with compoundsaccording to the present invention will reduce the likelihood or inhibita particular condition or disease state (in particular, the worsening ofa disease state such as the metastasis of cancer or other acceptedindicators of disease progression in the case of inflammatory andneurologic diseases) from occurring.

The term “cancer” shall refer to a proliferation of tumor cells havingthe unique trait of loss of normal controls, resulting in unregulatedgrowth, lack of differentiation, local tissue invasion, and/ormetastasis. As used herein, neoplasms include, without limitation,morphological irregularities in cells in tissue of a subject or host, aswell as pathologic proliferation of cells in tissue of a subject, ascompared with normal proliferation in the same type of tissue.Additionally, neoplasms include benign tumors and malignant tumors(e.g., colon tumors) that are either invasive or noninvasive. Malignantneoplasms are distinguished from benign neoplasms in that the formershow a greater degree of anaplasia, or loss of differentiation andorientation of cells, and have the properties of invasion andmetastasis. The term cancer also within context, includes drug resistantcancers, including multiple drug resistant cancers. Examples ofneoplasms or neoplasias from which the target cell of the presentinvention may be derived include, without limitation, carcinomas (e.g.,squamous-cell carcinomas, adenocarcinomas, hepatocellular carcinomas,and renal cell carcinomas), particularly those of the bladder, bone,bowel, breast, cervix, colon (colorectal), esophagus, head, kidney,liver, lung, nasopharyngeal, neck, ovary, pancreas, prostate, andstomach; leukemias, such as acute myelogenous leukemia, acutelymphocytic leukemia, acute promyclocytic leukemia (APL), acute T-celllymphoblastic leukemia, adult T-cell leukemia, basophilic leukemia,eosinophilic leukemia, granulocytic leukemia, hairy cell leukemia,leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia,lymphocytic leukemia, megakaryocytic leukemia, micromyeloblasticleukemia, monocytic leukemia, neutrophilic leukemia and stem cellleukemia; benign and malignant lymphomas, particularly Burkitt'slymphoma, Non-Hodgkin's lymphoma and B-cell lymphoma; benign andmalignant melanomas; myeloproliferative diseases; sarcomas, particularlyEwing's sarcoma, hemanglosarcoma, Kaposi's sarcoma, liposarcoma,myosarcomas, peripheral neuroepithelioma, and synovial sarcoma; tumorsof the central nervous system (e.g., gliomas, astrocytomas,oligodendrogliomas, ependymomas, gliobastomas, neuroblastomas,ganglioneuromas, gangliogliomas, medulloblastomas, pineal cell tumors,meningiomas, meningeal sarcomas, neurofibromas, and Schwannomas);germ-line tumors (e.g., bowel cancer, breast cancer, prostate cancer,cervical cancer, uterine cancer, lung cancer (e.g., small cell lungcancer, mixed small cell and non-small cell cancer, pleuralmesothelioma, including metastatic pleural mesothelioma small cell lungcancer and non-small cell lung cancer), ovarian cancer, testicularcancer, thyroid cancer, astrocytoma, esophageal cancer, pancreaticcancer, stomach cancer, liver cancer, colon cancer, and melanoma); mixedtypes of neoplasias, particularly carcinosarcoma and Hodgkin's disease;and tumors of mixed origin, such as Wilms' tumor and teratocarcinomas,among others. It is noted that certain epithelial tumors includingovarian, breast, colon, head and neck, medulloblastoma and B-celllymphoma, among others have all been shown to exhibit increased Rac andCdc42 expression or activation and are principal target cancers forcompounds and therapies according to the present invention.

The term “additional anti-cancer agent” is used to describe anadditional compound which may be coadministered with one or morecompounds of the present invention in the treatment of cancer. Suchagents include, for example, everolimus, trabectedin, abraxane, TLK 286,AV-299, DN-101, pazopanib, GSK690693, RTA 744, ON 0910.Na, AZD 6244(ARRY-142886), AMN-107, TKI-258, GSK461364, AZD 1152, enzastaurin,vandetanib, ARQ-197, MK-0457, MLN8054, PHA-739358, R-763, AT-9263, aFLT-3 inhibitor, a VEGFR inhibitor, an EGFR TK inhibitor, an aurorakinase inhibitor, a PIK-1 modulator, a Bcl-2 inhibitor, an HDACinhbitor, a c-MET inhibitor, a PARP inhibitor, a Cdk inhibitor, an EGFRTK inhibitor, an IGFR-TK inhibitor, an anti-HOF antibody, a PI3 kinaseinhibitors, an AKT inhibitor, a JAK/STAT inhibitor, a checkpoint-1 or 2inhibitor, a focal adhesion kinase inhibitor, a Map kinase kinase (mek)inhibitor, a VEOF trap antibody, pemetrexed, erlotinib, dasatanib,nilotinib, decatanib, panitumumab, amrubicin, oregovomab, Lep-etu,nolatrexed, azd2171, batabulin, ofatumumab, zanolimumab, edotecarin,tetrandrine, rubitecan, tesmilifene, oblimersen, ticilimumab,ipilimumab, gossypol, Bio 111, 131-I-TM-601, ALT-110, BIO 140, CC 8490,cilengitide, gimatecan, IL13-PE38QQR, INO 1001, IPdR₁ KRX-0402,lucanthone, LY 317615, neuradiab, vitespan, Rta 744, Sdx 102,talampanel, atrasentan, Xr 311, romidepsin, ADS-100380, sunitinib,5-fluorouracil, vorinostat, etoposide, gemcitabine, doxorubicin,irinotecan, liposomal doxorubicin, 5′-deoxy-5-fluorouridine,vincristine, temozolomide, ZK-304709, seliciclib; PD0325901, AZD-6244,capecitabine, L-Glutamic acid,N-[4-[2-(2-amino-4,7-dihydro-4-oxo-1H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-,disodium salt, heptahydrate, camptothecin, PEG-labeled irinotecan,tamoxifen, toremifene citrate, anastrazole, exemestane, letrozole,DES(diethylstilbestrol), estradiol, estrogen, conjugated estrogen,bevacizumab, IMC-1C11, CHIR-258);3-[5-(nmethylsulfonylpiperadinemethyl)-indolylj-quinolone, vatalanib,AG-013736, AVE-0005, the acetate salt of [D-Ser(But) 6, Azgly 10](pyro-Glu-His-Trp-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-Azgly-NH₂ acetate[C₅₉H₁₈N₁₈Oi₄-(C₂H₄O₂)_(x) where x=1 to 2.4], goserelin acetate,leuprolide acetate, triptorelin pamoate, medroxyprogesterone acetate,hydroxyprogesterone caproate, megestrol acetate, raloxifene,bicalutamide, flutamide, nilutamide, megestrol acetate, CP-724714;TAK-165, HKI-272, erlotinib, lapatanib, canertinib, ABX-EGF antibody,erbitux, EKB-569, PKI-166, GW-572016, lonafamib, BMS-214662, tipifamib;amifostine, NVP-LAQ824, suberoyl analide hydroxamic acid, valproic acid,trichostatin A, FK-228, SU11248, sorafenib, KRN951, aminoglutethimide,amsacrine, anagrelide, L-asparaginase, Bacillus Calmette-Guerin (BCG)vaccine, bleomycin, buserelin, busulfan, carboplatin, carmustine,chlorambucil, cisplatin, cladribine, clodronate, cyproterone,cytarabine, dacarbazine, dactinomycin, daunorubicin, diethylstilbestrol,epirubicin, fludarabine, fludrocortisone, fluoxymesterone, flutamide,gemcitabine, gleevac, hydroxyurea, idarubicin, ifosfamide, imatinib,leuprolide, levamisole, lomustine, mechlorethamine, melphalan,6-mercaptopurine, mesna, methotrexate, mitomycin, mitotane,mitoxantrone, nilutamide, octreotide, oxaliplatin, pamidronate,pentostatin, plicamycin, porfimer, procarbazine, raltitrexed, rituximab,streptozocin, teniposide, testosterone, thalidomide, thioguanine,thiotepa, tretinoin, vindesine, 13-cis-retinoic acid, phenylalaninemustard, uracil mustard, estramustine, altretamine, floxuridine,5-deooxyuridine, cytosine arabinoside, 6-mecaptopurine, deoxycoformycin,calcitriol, valrubicin, mithramycin, vinblastine, vinorelbine,topotecan, razoxin, marimastat, COL-3, neovastat, BMS-275291,squalamine, endostatin, SU5416, SU6668, EMD121974, interleukin-12,IM862, angiostatin, vitaxin, droloxifene, idoxyfene, spironolactone,finasteride, cimitidine, trastuzumab, denileukin diftitox, gefitinib,bortezimib, paclitaxel, irinotecan, topotecan, doxorubicin, docetaxel,vinorelbine, bevacizumab (monoclonal antibody) and erbitux,cremophor-free paclitaxel, epithilone B, BMS-247550, BMS-310705,droloxifene, 4-hydroxytamoxifen, pipendoxifene, ERA-923, arzoxifene,fulvestrant, acolbifene, lasofoxifene, idoxifene, TSE-424, HMR-3339,ZK186619, PTK787/ZK 222584, VX-745, PD 184352, rapamycin,40-O-(2-hydroxyethyl)-rapamycin, temsirolimus, AP-23573, RAD001,ABT-578, BC-210, LY294002, LY292223, LY292696, LY293684, LY293646,wortmannin, ZM336372, L-779, 450, PEG-filgrastim, darbepoetin,erythropoietin, granulocyte colony-stimulating factor, zolendronate,prednisone, cetuximab, granulocyte macrophage colony-stimulating factor,histrelin, pegylated interferon alfa-2a, interferon alfa-2a, pegylatedinterferon alfa-2b, interferon alfa-2b, azacitidine, PEG-L-asparaginase,lenalidomide, gemtuzumab, hydrocortisone, interleukin-1, dexrazoxane,alemtuzumab, all-transretinoic acid, ketoconazole, interleunkin-2,megestrol, immune globulin, nitrogen mustard, methylprednisolone,ibritgumomab tiuxetan, androgens, decitabine, hexamethylmelamine,bexarotene, tositumomab, arsenic trioxide, cortisone, editronate,mitotane, cyclosporine, liposomal daunorubicin, Edwina-asparaginase,strontium 89, casopitant, netupitant, an NK-1 receptor antagonists,palonosetron, aprepitant, diphenhydramine, hydroxyzine, metoclopramide,lorazepam, alprazolam, haloperidol, droperidol, dronabinol,dexamethasone, methylprednisolone, prochlorperazine, granisetron,ondansetron, dolasetron, tropisetron, pegfilgrastim, erythropoietin,epoetin alfa and darbepoetin alfa, among others.

The term “alkyl” is used herein to refer to a fully saturated monovalentradical containing carbon and hydrogen (up to 20 carbon atoms asotherwise indicated), and which may be a straight chain, branched orcyclic. Examples of alkyl groups are methyl, ethyl, n-butyl, n-heptyl,isopropyl, 2-methyl propyl, tert-butyl, neopentyl, etc.

The term “substituted” as that term relates to alkyl groups which aredescribed above include one or more functional groups such as loweralkyl groups containing 1-6 carbon atoms, aryl (phenyl or naphthyl),substituted aryl (as described below), acyl (C₁-C₆), halogen (F, Cl, Br,I, e.g., alkyl halos, e.g., CF₃), amido, thioamido, cyano, nitro,alkynyl (C₂-C₆), azido, hydroxy, alkoxy (C₁-C₆), amino, C₁-C₆ alkyl anddialkyl-amino, C₂-C₆ acylamino, C₂-C₆ oxyester or carboxyester, aryloxy,aryloxy(C₁-C₆)alkyl, carboxamido, thio, C₂-C₆ ether or thioether and thelike.

The term “aryl”, when used in context, refers to a substituted orunsubstituted monovalent aromatic radical having a single ring (e.g.,phenyl) or multiple condensed rings (e.g., naphthyl). Other examplesinclude heterocyclic aromatic (heteroaromatic or heteroaryl) ring groupshaving one or more nitrogen, oxygen, or sulfur atoms in the ring, suchas the 5 or 6-membered heteroaryls oxazolyl, isoxazolyl, pyrazolyl,thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, imidazolyl, furyl,pyrrolyl, pyridyl, thienyl, pyridazinyl, pyrimidyl, or the condensedphenyl/heteroaryl ring as otherwise described herein including, forexample, benzofuryl, benzothienyl, benzimidazolyl, benzotriazolyl,benzoxazolyl, benzothiazolyl, quinolyl, isoquinolyl and indolyl.

The term “substituted” as used in the term “substituted aryl,substituted aromatic, substituted heteroaryl, or substitutedheteroaromatic” herein signifies that one or more substituents (1, 2, 3or 4, preferably 1 or 2) may be present, said substituents beingselected from atoms and groups, which when present do not prevent thecompound from functioning as a modulator of GRPase. Examples ofsubstituents that may be present in a substituted aromatic orheteroaromatic group include, but are not limited to, groups such as(C₁-C₆) alkyl, (C₁-C₁₀), preferably, (C₂-C₆) acyl, aryl, heteroaryl,substituted aryl and heteroaryl, halogen, cyano, nitro, amido(optionally substituted with one or two C₁-C₆ alkyl groups), thioamido(optionally substituted with one or two C₁-C₅ alkyl groups), azido,alkynyl (C₂-C₆), (C₁-C₆) alkylhalos (e.g., CF₃), hydroxy, (C₁-C₆)alkoxy, (C₂-C₈) alkoxyalkyl, amino, (C₁-C₆) alkyl and dialkyl amino,(C₁-C₆ acylamino, (C₁-C₆) acyloxy, aryloxy, (C₁-C₆) aryloxyalkyl,(C₁-C₆) carboxyalkyl, carboxamido, thio, (C₁-C₆) thioethers, bothsaturated and unsaturated (C₃-C₈) cyclic hydrocarbons, (C₃-C₈)heterocycles and the like. It is noted that each of the substituentsdisclosed herein may themselves be substituted.

The term “heteroaryl” refers to an unsaturated carbocylic ring whereinone or more carbon atoms have been replaced with one or more heteroatomssuch as nitrogen, oxygen or sulfur. Examples of heteroaryls is describedabove. “5-membered heteroaryl” refers to heteroaryls containing 5 atomswithin the heteroaryl ring. “6-ring heteroaryls” refers to heteroarylscontaining 6 atoms within the heteroaryl ring. Heteroaryls may beunsubstituted or substituted as otherwise described herein. The term“heterocyclic” refers to a ring system containing from 3 to 8 atoms from1 to 4 of which are nitrogen, oxygen, or sulfur. 5- or 6-memberedheterocycles, when used, are preferred. Heterocycles may be saturated orunsaturated, depending upon the context of use. When unsaturatedheterocycles are also referred to as heteroaryls, when fully saturated,they are referred to as heterocycles.

The term “GTPase” is used to describe the Rho family of GTPases, whichis a family of small signaling GTPases, of which Rac1, Cdc42 and RhoAare the most well studied members. These GTPases have been shown toregulate many aspects of intracellular dynamics, and play a role in cellproliferation, apoptosis, gene expression, and multiple other commoncellular functions. They consequently have utility in the treatment ofsporadic and genetic diseases, as well as cancers as described herein.Rac1 is a GTPase regulator of a number of cellular processes, includingthe cell cycle, cell-cell adhesion, motility (through the actinnetwork), and of epithelial differentiation (for maintaining epidermalstem cells). Cdc42 is a GTPase protein involved in regulation of thecell cycle, cell differentiation and cell migration. RhoA is a GTPaseprotein which is involved in the regulation and timing of cell division.Together, these GTPase proteins are intimate to processes which arerelated to cancer and its elaboration and are targets for cancertreatment through modulation, in more particular aspects, inhibition ofthese GTPase targets. GTPase mediates a number of disease states,including cancer, as otherwise disclosed herein, as well as a number ofsporadic and genetic diseases including, for example, Menkes disease,rheumatoid arthritis, atherosclerosis, diabetes (type I), Huntington'sdisease and Alzheimer's disease, among others.

The term “Entamoeba” is used to describe a genus of protozoal anaerobicparasites found as internal parasites or commensals of animals.Entamoeba histolytica is an anaerobic parasitic protozoan, part of thegenus Entamoeba found in humans and responsible for diseases and/orconditions such as Amoebiasis; Amoebic dysentery; ExtraintestinalAmoebiasis, Amoebic Liver Abscess; Amoeba Cutis; and Amoebic LungAbscess (“liver-colored sputum”). E. histolytica predominantly infectshumans and other primates, as does E dlspar, although E. dispar isnon-pathogenic. Mammals such as dogs and cats can become infectedtransiently, but are not thought to contribute significantly totransmission. “Additional anti-entamoeba agents” are agents which may becombined with those of the present invention and used to inhibit andor/treat E. histolytica infections, disease states and/or conditions.These compounds include, for example, metronidazole, bismuthsubsalicylate, kaolin pectin, diphenoxyolate, loperamide, quinolones,erythromycin, trimethoprim-sulamethoxazole, ceftriaxine, ampicillin,tetracycline, doxycycline, vancomycin iodoquinol and mixtures thereof.“Acanthamoeba spp. is used to describe another genus of protozoalanaerobic parasites found as parasites or commensals of animals,including humans and is responsible for acanthamoebiasis of the eye(amoeba cutis acanthamoebiasis of the eye), which is also treated bycompounds according to the present invention.

The term “co-administration” or “adjunct therapy” shall mean that atleast two compounds or compositions are administered to the patient atthe same time, such that effective amounts or concentrations of each ofthe two or more compounds may be found in the patient at a given pointin time. Although compounds according to the present invention may beco-administered to a patient at the same time, the term embraces bothadministration of two or more agents at the same time or at differenttimes, including sequential administration. Preferably, effectiveconcentrations of all co-administered compounds or compositions arefound in the subject at a given time. The term co-administration oradjunct therapy also contemplates other bioactive agents beingcoadministered with pharmaceutical compositions according to the presentinvention, especially where a cancer has metastasized or is at risk formetastasis.

Compounds according to the present invention may be readily formulatedinto pharmaceutical compositions, useful in the treatment of sporadic orgenetic diseases or conditions, cancers or infections or conditionscaused by Entamoeba histolytica or Acanthamoieba spp. as describedhereinabove. Pharmaceutical compositions comprise an effective amount ofone or more compounds according to the present invention in combinationwith a pharmaceutically acceptable carrier, additive or excipient,optionally in combination with at least one additional anticancer agent.

The present invention includes the compositions comprising thepharmaceutically acceptable salt i.e., the acid or base addition saltsof compounds of the present invention and their derivatives. The acidswhich may be used to prepare the pharmaceutically acceptable acidaddition salts of the aforementioned base compounds useful in thisinvention are those which form non-toxic acid addition salts, i.e.,salts containing pharmacologically acceptable anions, such as thehydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate,phosphate, acid phosphate, acetate, lactate, citrate, acid citrate,tartrate, bitartrate, succinate, maleate, fumarate, gluconate,saccharate, benzoate, methanesulfonate, ethanesulfonate,benzenesulfonate, p-toluenesulfonate and pamoate [i.e.,1,1′-methylene-bis-(2-hydroxy-3 naphthoate)]salts, among others.

Pharmaceutically acceptable base addition salts may also be used toproduce pharmaceutically acceptable salt forms of the compoundsaccording to the present invention. The chemical bases that may be usedas reagents to prepare pharmaceutically acceptable base salts of thepresent compounds that are acidic in nature are those that formnon-toxic base salts with such compounds. Such non-toxic base saltsinclude, but are not limited to those derived from suchpharmacologically acceptable cations such as alkali metal cations (eg.,potassium and sodium) and alkaline earth metal cations (e, calcium andmagnesium), ammonium or water-soluble amine addition salts such asN-methylglucamine-(meglumine), and the lower alkanolammonium and otherbase salts of pharmaceutically acceptable organic amines, among others.

As noted above, the compounds and method of the invention modulateGTPase as otherwise described herein, and are useful for the inhibition(including prophylaxis) and/or treatment of cancer, sporadic or geneticdiseases or conditions and infections caused by Entamoeba sp., includingE. histolytica, and other amoeba species responsible for amoebicdysentery as well as other infections, e.g., acanthamoebiasis of the eyewhich is caused by Acanthamoeba.

In methods according to the present invention, subjects or patients inneed are treated with the present compounds, pharmaceutical compositionsin order to inhibit, reduce the likelihood or treat a disease state,condition and/or infection as otherwise described herein. The diseasestates, conditions and infections treated by the present compounds andcompositions are readily recognized and diagnosed by those of ordinaryskill in the art and treated by administering to the patient aneffective amount of one or more compounds according to the presentinvention.

Generally, dosages and routes of administration of the compound aredetermined according to the size and condition of the subject, accordingto standard pharmaceutical practices. Dose levels employed can varywidely, and can readily be determined by those of skill in the art.Typically, amounts in the milligram up to gram quantities are employed.The composition may be administered to a subject by various routes, e.g.orally, transdermally, perineurally or parenterally, that is, byintravenous, subcutaneous, intraperitoneal, or intramuscular injection,among others, including buccal, rectal and transdermal administration.Subjects contemplated for treatment according to the method of theinvention include humans, companion animals, laboratory animals, and thelike.

Formulations containing the compounds according to the present inventionmay take the form of solid, semi-solid, lyophilized powder, or liquiddosage forms, such as, for example, tablets, capsules, powders,sustained-release formulations, solutions, suspensions, emulsions,suppositories, creams, ointments, lotions, aerosols, patches or thelike, preferably in unit dosage forms suitable for simple administrationof precise dosages.

Pharmaceutical compositions according to the present invention typicallyinclude a conventional pharmaceutical carrier or excipient and mayadditionally include other medicinal agents, carriers, adjuvants,additives and the like. Preferably, the composition is about 0.1% toabout 85%, about 0.5% to about 75% by weight of a compound or compoundsof the invention, with the remainder consisting essentially of suitablepharmaceutical excipients. For oral administration, such excipientsinclude pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharine, talcum, cellulose, glucose, gelatin,sucrose, magnesium carbonate, and the like. If desired, the compositionmay also contain minor amounts of non-toxic auxiliary substances such aswetting agents, emulsifying agents, or buffers.

Liquid compositions can be prepared by dissolving or dispersing thecompounds (about 0.5% to about 20% by weight or more), and optionalpharmaceutical adjuvants, in a carrier, such as, for example, aqueoussaline, aqueous dextrose, glycerol, or ethanol, to form a solution orsuspension. For use in oral liquid preparation, the composition may beprepared as a solution, suspension, emulsion, or syrup, being suppliedeither in liquid form or a dried form suitable for hydration in water ornormal saline.

When the composition is employed in the form of solid preparations fororal administration, the preparations may be tablets, granules, powders,capsules or the like. In a tablet formulation, the composition istypically formulated with additives, e.g. an excipient such as asaccharide or cellulose preparation, a binder such as starch paste ormethyl cellulose, a filler, a disintegrator, and other additivestypically used in the manufacture of medical preparations.

An injectable composition for parenteral administration will typicallycontain the compound in a suitable i.v. solution, such as sterilephysiological salt solution. The composition may also be formulated as asuspension in a lipid or phospholipid, in a liposomal suspension, or inan aqueous emulsion.

Methods for preparing such dosage forms are known or are apparent tothose skilled in the art; for example, see Remington's PharmaceuticalSciences (17th Ed., Mack Pub. Co., 1985). The composition to beadministered will contain a quantity of the selected compound in apharmaceutically effective amount for modulating GTPase in a subjectaccording to the present invention in a subject.

Synthesis of Compounds According to the Present Invention

The compounds according to the present invention are readily known inthe art and their synthesis is well-known and readily provided. Most ofthe compounds are known in the art and can be found in publishedliterature, purchased from commercial sources or readily prepared fromstarting materials which are readily obtained from commercial sources.Substituted phenyl or naphthyl compounds, principally used in thepresent invention are well known in the art. The various substituentsmay be readily introduced into the pharmacophore, whether thatpharmacophore is a phenyl group to which substituents as otherwisepresented herein are introduced, a naphthyl group as otherwise describedherein or a fused bicyclic benzoheteroaryl group as described herein.Synthesis of all of the presently described compounds are well withinthe routineer's skill in the art.

Method of Treatment

According to one aspect of the invention, a method is provided fortreating a mammalian patient or subject to modulate GTPase, inparticular the Rho family of GTPases including Rac (e.g. Rac1), Cdc42and Rho (e.g. Rho1). Agonist and/or antagonist activity of compoundsaccording to the present invention described herein may be used tomodulate GTPase in a manner consistent with inhibiting and/or treatingdisease states and/or conditions including cancer, sporadic or geneticdiseases including Menkes disease, rheumatoid arthritis,atherosclerosis, diabetes (type I), Huntington's disease and Alzheimer'sdisease, as otherwise described herein and infections caused byEntamoeba histolytica or Acanthamoeba spp. Antagonist activityassociated with GTPase inhibition is a particularly useful aspect of thepresent invention.

According to the present invention, in patients or subjects in needthereof, are treated by administering to the patient or subject aneffective amount of one or more compounds according to the presentinvention, optionally in combination with at least one additionalbioactive agent useful for treating the same disease state or condition.Compounds according to the present invention may be used to inhibit,reduce the likelihood or treat cancer, including the metastasis ofcancer in a patient or subject in need of such treatment. The treatmentis useful for any cancer which is mediated by GTPase or for whichmetastasis is a risk element. Therapy with at least one additionalanticancer agent as otherwise described herein is also contemplated inthe present methods. The numerous cancers which may be treated pursuantto the present method is described hereinabove.

In another aspect the present invention is directed to a method fortreating a sporadic or genetic disease in which activation of Rho GTPaseplays a significant role. These disease states and/or conditionsinclude, for example Menkes disease, rheumatoid arthritis,atherosclerosis, diabetes (type I), Huntington's disease and Alzheimer'sdisease. In this method, a patient or subject in need of treatment isadministered an effective amount of a compound as otherwise describedherein optionally in combination with a pharmaceutically acceptablecarrier, additive or excipient.

In another aspect, the invention provides a method for reducing,inhibiting and/or treating infections, disease states or conditionswhich are caused by Entamoeba histolytica. These disease states and/orconditions include amoebiasis, amoebic dysentery, extraintestinalamoebiasis, amoebic liver abscess, amoeba cutis, and amoebic lungabscess. In this aspect, the method of the present invention comprisesadministering to a subject or patient in need an amount of a compound ofthe invention, the amount being sufficient to reduce, inhibit or cureinfection and/or a disease state and/or condition caused by Entamoebahistolytica or Acanthamoeba spp. The compounds according to the presentinvention may be used alone or combined with another agent useful intreating infections caused by Entamoeba histolytica or Acanthamoeba,including metronidazole, bismuth subsalicylate, kaolin pectin,diphenoxyolate, loperamide, quinolones, erythromycin,trimethoprim-sulamethoxazole, cefIriaxine, ampicillin, tetracycline,doxycycline, vancomycin iodoquinol and mixtures thereof.

In the present invention, the method of treatment comprisesadministering to the subject in need of treatment, in a pharmaceuticallyacceptable carrier, an effective amount of a compound according to Ibelow:

Wherein R¹ and R² are each independently H or a C₁-C₃ alkyl group;

R³ is a

group,

where Y is absent, O or S;

n is 0, 1, 2, 3, 4, 5 or 6;

R is H, a C₁-C₂₀ alkyl group or a phenyl group optionally substitutedwith a hydroxyl, halo or C₁-C₃ alkyl group;

R^(1a) and R^(1b) are each independently H or a CH₃ group with theproviso that at least one of R^(1a) and R^(1b) is a methyl group; and

said —(CH₂)_(n)— moiety is optionally substituted with a halo group (F,Cl, Br or I), a C₁-C₃ alkyl group or a hydroxyl group (preferably, themethylene group alpha to the carbon group containing the R^(1a) andR^(1b) substituents is substituted with a methyl or ethyl group);

R⁴ is H or a C₁ to C₄ alkyl group;

R⁵ is H, halo (F, Cl, Br or I), a C₁-C₆ linear or branch-chained alkylgroup (preferably a C₁-C₄alkyl group), a C₁-C₆ alkoxy group or a

group where R₅ is an optionally substituted phenyl or a 5- or 6-memberedheteroaryl group, or R⁵, together with R⁶, forms an optionallysubstituted phenyl group (preferably a C₁-C₄ alkoxy, e.g. methoxy orethoxy substituted phenyl group) or an optionally substituted 5- or6-membered heteroaryl ring (preferably, an oxazole ring, preferablysubstituted with a ortho- or para-halogen e.g. F, Cl, substituted phenylgroup) thus forming a bicyclic ring system; andR⁶ is H, halo, C₁-C₆ linear or branch-alkyl group (preferably, a C₁-C₄alkyl group), a C₁-C₆ alkoxy group or a

group where R₅ is an optionally substituted phenyl or a 5- or 6-memberedheteroaryl group, or R⁶, together with R⁵, forms an optionallysubstituted phenyl group or an optionally substituted 5- or 6-memberedheteroaryl ring, thus forming an optionally substituted bicyclic ringsystem, ora pharmaceutically acceptable salt, enantiomer, solvate or polymorphthereof.

In preferred aspects of the invention, R⁵ and R⁶ together form a phenylgroup which is optionally substituted by at least one methoxy group(preferably, furthermost from the R³ substituent as in naproxen,methallenestril and flunoxaprofen—see FIG. 10 hereof) and R⁴ is H. Inother aspects of the invention, R^(1a) is methyl and R^(1b) is hydrogenproviding a chiral center and the possibility of racemic mixtures andindividual enantiomers, each of which may be used in the presentinvention. In substituent R³, n is preferably 0 or 1 and Y is absent. Inalternative preferred embodiments, when R⁵ and R⁶ together form a phenylor hetetoaryl group, in the R³ substitutent, Y is absent and n is 0 or 1and when n is 1, the methylene group is substituted with a C₁-C₃ alkyl(preferably ethyl) and R^(1a) and R^(1b) are both methyl. When n is 0,only one of R_(1a) and R_(1b) is H, thus forming a chiral center. Inalternative aspects of the invention where R⁵ and R⁶ do not form a ringto create a bicyclic group, Y in R³ is preferably 0 or absent, n is 0,1, 2, 3, or 4 (preferably 0 or 3), R^(1a) is methyl and R^(1b) is H(thus forming a chiral center). In these monocyclic embodiments, R⁵ is Hor a halogen group, preferably H, R⁶ is H or a C₁-C₆ alkyl group(preferably H or a C₃ or C₄ linear or branch-chained alkyl group, R⁴ ispreferably H and R¹ and R² are H or CH₃, preferably H.

The alternative embodiments, the present invention relates to methods oftreatment wherein the compound is selected from the group consisting ofR-Naproxen, S-Naproxen, methallenestril, R-Flunoxaprofen,S-flunoxaprofen, R-Ibuprofen, S-Ibuprofen, S-Ketoprofen, R-Ketoprofen,gemfibrozil, ecabet, exetecan acid, R-Ketoralac, S-Ketoralac,tanomastat, mitiglinide, cicloxillic acid, fexofenadine, cilomilast,levocabastine, tiagabine, cinalukrast and mixtures thereof; includingpharmaceutically acceptable salts, enantiomers, racemic mixtures,solvates and polymorphs thereof. A number of these compounds or theirsalts are presented in attached FIG. 10.

In the methods treating or inhibiting cancer or the metastasis ofcancer, the compounds described above may be coadministered with atleast one additional anticancer agent including, for example,everolimus, trabectedin, abraxane, TLK 286, AV-299, DN-101, pazopanib,GSK690693, RTA 744, ON 0910.Na, AZD 6244 (ARRY-142886), AMN-107,TKI-258, GSK461364, AZD 1152, enzastaurin, vandetanib, ARQ-197, MK-0457,MLN8054, PHA-739358, R-763, AT-9263, a FLT-3 inhibitor, a VEGFRinhibitor, an EGFR TK inhibitor, an aurora kinase inhibitor, a PIK-1modulator, a Bcl-2 inhibitor, an HDAC inhbitor, a c-MET inhibitor, aPARP inhibitor, a Cdk inhibitor, an EGFR TK inhibitor, an IGFR-TKinhibitor, an anti-HGF antibody, a PI3 kinase inhibitors, an AKTinhibitor, a JAK/STAT inhibitor, a checkpoint-1 or 2 inhibitor, a focaladhesion kinase inhibitor, a Map kinase kinase (mek) inhibitor, a VEGFtrap antibody, pemetrexed, erlotinib, dasatanib, nilotinib, decatanib,panitumumab, amrubicin, oregovomab, Lep-etu, nolatrexed, azd2171,batabulin, ofatumumab, zanolimumab, edotecarin, tetrandrine, rubitecan,tesmilifene, oblimersen, ticilimumab, ipilimumab, gossypol, Bio 111,131-I-TM-601, ALT-110, BIO 140, CC 8490, cilengitide, gimatecan,IL13-PE38QQR, INO 1001, IPdR₁ KRX-0402, lucanthone, LY 317615,neuradiab, vitespan, Rta 744, Sdx 102, talampanel, atrasentan, Xr 311,romidepsin, ADS-100380, sunitinib, 5-fluorouracil, vorinostat,etoposide, gemcitabine, doxorubicin, irinotecan, liposomal doxorubicin,5′-deoxy-5-fluorouridine, vincristine, temozolomide, ZK-304709,seliciclib; PD0325901, AZD-6244, capecitabine, L-Glutamic acid,N-[4-[2-(2-amino-4,7-dihydro-4-oxo-1H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-,disodium salt, heptahydrate, camptothecin, PEG-labeled irinotecan,tamoxifen, toremifene citrate, anastrazole, exemestane, letrozole,DES(diethylstilbestrol), estradiol, estrogen, conjugated estrogen,bevacizumab, IMC-1C11, CHIR-258);3-[5-(methylsulfonylpiperadinemethyl)-indolylj-quinolone, vatalanib,AG-013736, AVE-0005, the acetate salt of [D-Ser(But) 6, Azgly 10](pyro-Glu-His-Trp-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-Azgly-NH₂ acetate[C₅₉H₈₄N₁₈Oi₄-(C₂H₄O₂)_(x) where x=1 to 2.4], goserelin acetate,leuprolide acetate, triptorelin pamoate, medroxyprogesterone acetate,hydroxyprogesterone caproate, megestrol acetate, raloxifene,bicalutamide, flutamide, nilutamide, megestrol acetate, CP-724714;TAK-165, HKI-272, erlotinib, lapatanib, canertinib, ABX-EGF antibody,erbitux, EKB-569, PKI-166, GW-572016, lonafarnib, BMS-214662,tipifarnib; amifostine, NVP-LAQ824, suberoyl analide hydroxamic acid,valproic acid, trichostatin A, FK-228, SU11248, sorafenib, KRN951,aminoglutethimide, arnsacrine, anagrelide, L-asparaginase, BacillusCalmette-Guerin (BCG) vaccine, bleomycin, buserelin, busulfan,carboplatin, carmustine, chlorambucil, cisplatin, cladribine,clodronate, cyproterone, cytarabine, dacarbazine, dactinonycin,daunorubicin, diethylstilbestrol, epirubicin, fludarabine,fludrocortisone, fluoxymnsterone, flutamide, gemcitabine, gleevac,hydroxyurea, idarubicin, ifosfamide, imatinib, leuprolide, levamisole,lomustine, mechlorethamine, melphalan, 6-mercaptopurine, mesna,methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, octreotide,oxaliplatin, pamidronate, pentostatin, plicamycin, porfimer,procarbazine, raltitrexed, rituximab, streptozocin, teniposide,testosterone, thalidomide, thioguanine, thiotepa, tratinoin, vindesine,13-cis-retinoic acid, phenylalanine mustard, uracil mustard,estramustine, altretamine, floxuridine, 5-dcooxyuridine, cytosinearabinoside, 6-mecaptopurine, deoxycoformycin, calcitriol, valrubicin,mithramycin, vinblastine, vinorelbine, topotecan, razoxin, marimastat,COL-3, neovastat, BMS-275291, squalamine, endostatin, SU5416, SU6668,EMD121974, interleukin-12, IM862, angiostatin, vitaxin, droloxifene,idoxyfene, spironolactone, finasteride, cimitidine, trastuzumab,denileukin diftitox, gefitinib, bortezimib, paclitaxel, irinotecan,topotecan, doxorubicin, docetaxel, vinorelbine, bevacizumab (monoclonalantibody) and erbitux, cremophor-free paclitaxel, epithilone B,BMS-247550, BMS-310705, droloxifene, 4-hydroxytamoxifen, pipendoxifene,ERA-923, arzoxifene, fulvestrant, acolbifene, lasofoxifene, idoxifene,TSE-424, HMR-3339, ZK186619, PTK787/ZK 222584, VX-745, PD 184352,rapamycin, 40-O-(2-hydroxyethyl)-rapamycin, temsirolimus, AP-23573,RAD001, ABT-578, BC-210, LY294002, LY292223, LY292696, LY293684,LY293646, wortmannin, ZM336372, L-779,450, PEO-filgrastim, darbepoetin,crythropoietin, granulocyte colony-stimulating factor, zolendronate,prednisone, cetuximab, granulocyte macrophage colony-stimulating factor,histrelin, pegylated interferon alfa-2a, interferon alfa-2a, pegylatedinterferon alfa-2b, interferon alfa-2b, azacitidine, PEO-L-asparaginase,lenalidomide, gemtuzumab, hydrocortisone, interleukin-11, dexrazoxane,alemtuzumab, all-transretinoic acid, ketoconazole, interleukin-2,megestrol, immune globulin, nitrogen mustard, methylprednisolone,ibritgumomab tiuxetan, androgens, decitabine, hexamethylmelamine,bexarotene, tositumomab, arsenic trioxide, cortisone, editronate,mitotane, cyclosporine, liposomal daunorubicin, Edwina-asparaginase,strontium 89, casopitant, netupitant, an NK-1 receptor antagonists,palonosetron, aprepitant, diphenhydramine, hydroxyzine, metoclopramide,lorazepam, alprazolam, haloperidol, droperidol, dronabinol,dexamethasone, methylprednisolone, prochlorperazine, granisetron,ondansetron, dolasetron, tropisetron, pegfilgrastim, erythropoietin,epoetin alfa and darbepoetin alfa, among others, and mixtures thereof.

In methods involving infections, disease states and/or conditions causedby Entamoeba histolyltica or Acanthamoeba, including amoebiasis, amoebicdysentery, extraintestinal amoebiasis, amoebic liver abscess, amoebacutis, amoebic lung abscess and acanthmoebiasis of the eye(Acanthamoebia), at least one compound according to the presentinvention, alone or in combination with at least one further agentselected from the group consisting of metronidazole, bismuthsubsalicylate, kaolin pectin, diphenoxyolate, loperamide, quinolones,erythromycin, trimethoprim-sulamethoxazole, ceftriaxine, ampicillin,tetracycline, doxycycline, vancomycin iodoquinol and mixtures thereof isadministered in an effective amount to a patient of subject in needthereof.

The following examples illustrate but are not intended in any way tolimit the invention.

Experimental Rationale and Approach

The Ras-homologous (Rho) family of small GTPases (Rac, Cdc42 and Rho)are key regulators of actin reorganization, cell motility, cell-cell andcell-extracellular matrix (ECM) adhesion as well as of cell cycleprogression, gene expression and apoptosis (FIG. 1) [1-8]. In many humancancers (including colon and breast), aberrant Rho-family signaling dueto changes in the GTPase itself or in its regulation loops is a criticalunderpinning of tumor growth and survival, invasion and metastasis[9-13] (FIG. 1). RhoA and RhoC correlate with advanced ovarian cancerand peritoneal dissemination [14; 15]. Our own studies are the first todemonstrate dysregulation of Rac1 and Cdc42 in ovarian tumor specimens(FIG. 2).

Although Rac1 and Cdc42 have been recognized as attractive therapeutictargets, specific Rac GTPase inhibitors while effective in culture [16;17] have not been translated to clinical use and there are noestablished Cdc42 specific inhibitors. Lovastatin was shown to inhibitRho GTPase and reduce ovarian metastasis in a xenograft model [15].However, the use of statins to block GTPase membrane association has metwith only modest success due to their broad spectrum inhibition ofprotein prenylation resulting in pleiotropic effects on many GTPases andpathways [18]. Thus, more specific agents for clinical application areurgently needed. The inventors identified two FDA approved drugs asinhibitors of Rac and Cdc42 that block ovarian cancer cell behaviorsassociated with tumor growth, dissemination and invasion (FIGS. 3-4) anddisplay activity an intraperitoneal xenograft model (FIG. 6).

The studies described herein are significant for several reasons. First,the inventors investigate Rac1 and Cdc42 as therapeutic targets inovarian cancer, a disease with limited treatment options. Effectivetargeting of key regulators of metastatic spread would have substantialpositive impact on patient quality of life and duration of disease-freesurvival. Second, the inventors determine the mechanism of action of thenovel Rac1 and Cdc42 inhibitors that will inform future development oftargeted therapeutics. Third, testing FDA approved drugs for impact onovarian cancer enable positive results to be rapidly translated to phase1/2 clinical trials. Finally, results are expected to be applicable tonumerous other cancers where Rac and Cdc42 and their downstreameffectors are aberrantly activated.

Other disease states and conditions which are favorably impacted by theresults which are obtained from the experiments described herein includethe sporadic or genetic disease states and/or conditions in which RhoGTPases are implicated including, for example, Menkes disease,rheumatoid arthritis, atherosclerosis, diabetes (type I), Huntington'sdisease and Alzheimer's disease. Inhibition of GTPase is consistent withtreatment modalities for these disease states and/or conditions whichmediated through Rho GTPase.

High Throughput Screens

A first task involved conducting comprehensive high throughput screensfor inhibitors and activators of small GTPases using purified proteinsand libraries of both FDA approved and novel small molecules [19; 20].Cheminformatics identified structure-activity relationships that allowedgeneration of testable, mechanistic hypotheses for Rac1 and Cdc42inhibition by stereoisomeric compounds, including naproxen andketorolac. The lack of activity by >20 other NSAIDs against GTPasetargets strongly suggests enantiomer-selective targeting of Rac1 andCdc42 by R-naproxen and R-ketorolac and related derivatives (FIG. 10).Although it has been long recognized that R-enantiomers of NSAIDS arepoor inhibitors of COX activity and lack COX-associated toxicities[21-29], little is known about potential pharmacologic activities ortargets of the R-enantiomers. In one example, R-etodolac has no COXinhibitory activity, but does retard tumor development and metastasis ina transgenic mouse model of prostate cancer [30]. The retinoid Xreceptor-α was identified as the R-etodolac target, indicatingenantiomer-dependent selectivity for a novel target.

The discovery of the presently claimed compounds is based on highthroughput screening, cheminformatics and direct biochemical andcellular testing showing that R-naproxen and R-ketorolac inhibit Rac1and Cdc42 GTPase activities. Despite substantial evidence for a causalrole of aberrant Rac1 and/or Cdc42 activity in human cancers, theseproteins have not been widely explored as therapeutic targets. Thelimited numbers of small molecule Rac inhibitors (NSC23766 andderivatives, EHT 1864) disrupt specific activator proteins (i.e. GEFs)that convert the GTPase to the active state, or perturb effectorcoupling to block downstream signaling (FIG. 1) [16; 31-33].

Cheminformatic modeling of R-naproxen binding to Rac1 predicts thecompound docks to an allosteric site in the nucleotide-binding pocket ofthe GDP-bound GTPase; suggesting a novel mechanism of action that isdistinct from the current Rac inhibitors (FIG. 3) and the proposedstudies may reveal a new avenue for disrupting Rac and Cdc42 activity.Although Rac1-targeted inhibitors display activity in cells andrepresent promising leads [16; 17; 33; 34], these compounds have notbeen translated to human use and Cdc42-specific inhibitors are notreported in the literature. In one aspect of the invention, therepurposing of FDA-approved drugs would offer a distinct advantage overnew chemical entities requiring extensive toxicity and safety testingprior to clinical application. Thus, the R-enantiomers of naproxen andketorolac (FDA approved in the racenic mixtures) and compounds listed inFIG. 10 offer an innovative approach for therapeutic targeting of Rac1and Cdc42. The proposed studies provide critical information on themechanism and feasibility of targeting Rac1 and Cdc42 with theR-enantiomers of naproxen and ketorolac. The expertise of themulti-disciplinary investigative team and collaborators(cheminformatics, bioinformatics, crystallography, biochemistry, cellbiology and in vivo tumor modeling) fosters the advancement of theseRac1/Cdc42 targeted compounds with potential for rapid translation, andconversely validate Rac and Cdc42 as novel therapeutic targets inovarian cancer.

Rationale and Feasibility of the Examples:

Ovarian cancer metastasis is predicted to be strongly dependent onRac1/Cdc42-regulated pathways for exfoliation, formation ofmulticellular aggregates (MCAs), mesothelial adhesion, and localizedinvasion into the interstitial collagen-rich submesothelial matrix(FIG. 1) [35-38]. The preliminary data generated offer the firstevidence for dysregulation of Rac1 and Cdc42 GTPase expression andactivity in ovarian cancer (FIG. 2). Stage and grade dependentoverexpression of Cdc42 and Rac1 proteins was based onimmunohistochemical staining of human tumor samples. GTPaseoverexpression levels were highly significant for malignant, high-gradetumors (Rac1 (p=0.009) and Cdc42 (p<0.001)). Expression of aconstitutively active splice variant Rac1b (first documented in breasttumors, [12]) was detected by qPCR and a GLISA activity assay showedboth Cdc42 and Rac1 GTPases highly active in fresh tumor isolates. Thesedata provide the foundation for further experiments which are providedto delineate the impact of Rac1 and Cdc42 on measures of ovarian cancerdisease and patient outcomes. Because activated Rac1 and Cdc42 regulatecell motility, survival and proliferation through multiple effectors(FIG. 1), we also investigate the status of effector proteins associatedwith the tumor cell behaviors that are altered by Rac1 and Cdc42inhibitors (Table 1, FIG. 4). This information delivers mechanisticinsight into the contributions of Rac1 and Cdc42 pathways to ovariancancer and identify key downstream effector pathways associated withthis disease. Access to a repository of tumor samples that are linked topatient outcomes through the Human Tissue Repository of the UNM NCIfunded Cancer Center is important to deceipher these pathways.

Further Examples

Screening of the Prestwick library of FDA-approved molecules identifiedthe R-enantiomer of naproxen as active against Rac1 and Cdc42, which wasconfirmed in cellular GLISA assays (FIG. 3A). R-, and S-ketorolac wereidentified for structure activity testing based on cheminformatics.Despite the common chemotype, S-naproxen and 6-MNA did not significantlyinhibit Rac1 or Cdc42 indicating that GTPase inhibition is independentof the COX pathway. At nontoxic doses that are meaningful with respectto human serum levels at therapeutic doses Table 2 [39-47], theR-enantiomers of naproxen or ketorolac are more effective for inhibitionof migration in SKOV3ip (FIG. 4A-B), OVCA 429 (not shown) and primaryovarian tumor cells isolated from ascites (not shown). The samestructure activity relationship was observed for multicellular aggregateformation (FIG. 4C). Inhibition of proliferation did not exceed 50% withR-naproxen>S-naproxen or 6-MNA and little inhibition by R- orS-ketorolac (not shown). We tested >20 other common NSAIDS and did notdetect inhibitory activity in vitro or in cells against Rac1 or Cdc42,nor any impact on ovarian cell migration or proliferation. Thus, fromthe structure activity relationships and selectivity, we conclude thatthe inhibitory actions of R-naproxen and R-ketorolac are due to aneffect on novel Rac1 and Cdc42 GTPase targets as first revealed by themolecular library screen and predicted by cheminformatics.

Virtual docking predicts that R-naproxen, but not S-naproxen can bindthe GDP-bound pocket of Rac1 (FIG. 3B). In the model R-naproxen isstabilized through favorable H-bonds with Thr17 and Asp57, as well asthrough interaction with magnesium via the naproxen carboxyl group.Interestingly, Thr17 is the mutated residue in dominant negative formsof Rac1. The model predicts stabilization of the inactive GDP-boundGTPase and provides a testable hypothesis for a novel mechanism ofaction and the enantiomer selective differences in activity. Aspredicted by the enantiomer-selective inhibition of Rac1 and Cdc42activity, we find that R-naproxen and the established Rac1 inhibitorNSC23766 inhibit ovarian tumor cell proliferation, migration and MCAformation (FIGS. 4A-C and data not shown) while S-naproxen and 6-MNAdisplay little effect. Similarly, in an intraperitoneal xenograft model,animals administered R-naproxen displayed a 4-fold reduction in tumornumber and a 36% decrease in total tumor burden while the S-naproxen and6-MNA treated animals were similar to placebo controls (FIG. 6). Thesedata demonstrate that the enantiomer-selective properties of R-naproxenare preserved in vivo.

Establishing the Consequences of Rac1 and Cdc42 Activation andOverexpression in Human Ovarian Tumors.

Preliminary data generated represent the first evidence for increasedCdc42 and Rac1 expression and activation in ovarian cancer, and supportthe hypothesis that dysregulated Rac1 and Cdc42 will lead to theactivation of one or more effectors and pathways that have negativeimpact on ovarian cancer patient outcome. Because several effectors arereportedly elevated or activated in ovarian cancer, it is important toaddress whether effector activation is dependent or independent of Rac1and Cdc42 dysregulation to gain insights into the potential therapeuticimpact of targeting Rac1 and Cdc42. No such integrated analyses havebeen conducted in ovarian cancer or in other cancers where Rho-familyGTPases are overexpressed.

Focus is on the four downstream effectors (Table 1) with key roles intumor growth and metastasis (FIG. 1). In human cancers, p21 activatedkinase (PAK) isoforms act as nuclear effectors and are critical fortumor angiogenesis, epithelial-mesenchymal transition and anchorageindependent growth downstream of Rac1 and Cdc42 [36; 48]. Elevated PAK1levels are correlated with poor prognosis and aggressive ovariancancers, making it an effector of particular interest [49; 50].Cell-cell adhesion is modulated by active Rac1 and Cdc42 throughIQGAP1-mediated disassembly of adherens junctions and nucleartranslocation of beta catenin [38]. Rac/Cdc42/IQGAP1 are predicted tomodify MCA formation and/or dispersal upon mesothelial contact.Mesothelial invasion and contact with the collagen-rich submesothelialmatrix is likely mediated through WAVE3 and WASP effectors, whichfunction in actin remodeling and invasion. Activated Rac1 is importantfor integrin-mediated lamellipodia formation, cell spreading, and tumorcell migration via WAVE3 [51-53], while Cdc42 is important forinvadopodia-mediated invasion via WASP [37]. WAVE3 and WASP pathways aredownstream of activated Rac1 and Cd42, respectively, and is importantfor discriminating if one or both GTPase pathways are active, andidentify if functional redundancies amplify invasion or if one pathwaypredominates over another. The initial focus on these specific effectorsis based on the cellular functions disrupted by R-naproxen, R-ketorolacand the Rac1 inhibitor NSC23766 (FIGS. 4-5) and tumor cell activitiesthat are necessary for ovarian cancer metastatic success. Experimentsare designed to test the hypothesis that comparative evaluation of acohort of interrelated signaling molecules in patient tissue sampleswill demonstrate their utility individually or as a panel for serving asprognostic markers and provide supporting evidence for their use astherapeutic targets.

General Approach to Analysis:

Immunohistochemical (IHC) analysis of ovarian tumor tissue microarrays(TMAs) is conducted using commercially available sources andUNM-generated TMAs with associated patient information. IHC images arerecorded using an Aperio system with options for training and automatedquantification. Independent verification of tumor pathology and markerevaluation is conducted by Ob/Gyn certified pathologist Lomo andstatistical analysis is performed by the UNM CRTC biostatistics core asin FIG. 2.

Pathway Activation:

Stain for the markers indicated above are performed using commerciallyavailable tumor microarrays with multiple cores per case (US Biomax).Marker expression and localization (nuclear, cytoplasmic, membrane) isassessed. For example, PAK1 activation through phosphorylation andnuclear translocation is postulated to be a prognostic marker in ovariancancer based on its association with poor overall and disease-freesurvival and activation of angliogenesis [54] therefore localization isan important indicator of activation status. Scoring is conducted by twopathologists for the presence or absence of staining, localization andgood correlation (within 1 quartile) with percent and intensity (0-3+)of epithelial staining. Staining intensity is dichotomized with high- oroverexpression defined as above the median of the product of intensitytimes the percentage of epithelial cells stained. There are two levelsof statistical analysis. First, the protein expression levels and/ordistribution of each marker is analyzed with respect to tumor stage andgrade and ovarian carcinoma histotypes (serous, clear cell,endometrioid, and mucinous). This study will identify markers with thegreatest correlation to tumor parameters (histotype and diseasestage/grade). The second analysis is to evaluate PAK1/pPAK1, WAVE3, WASPand IQGAP1 protein expression levels and distribution as functions ofRac1 and/or Cdc42 activation or overexpression in serial sections. Theseexperiments will identify effectors with the greatest correlation toGTPase activation status and whether GTPase activation and specificdownstream pathways are coordinately activated.

Patient Outcome

Rac1, Cdc42 and downstream effectors identified as being linked toovarian tumor stage or grade are further tested on tumor microarrayswith linked patient outcome data. A duplicate core tissue microarray(TMA) of archival specimens from 45 patients with ovarian tumors of lowmalignant potential (LMP) and 89 patients with epithelial ovarian cancer(EOC) are used. This TMA was used to identify the novel estrogenreceptor GPR30 as a predictor of poor survival in ovarian cancer [55].Based on ANOVA analyses and 45% differences in expression levels, apower of 0.8 is achieved through the evaluation of 80 samples, which iswithin our sample collection. Marker expression or localization, definedas above or below the median (intensity in compartment×the percentage ofpositive epithelial cells), is used to correlate staining withpredictors of adverse outcome and overall or disease free survival.Additional arrays are prepared as needed. These experiments are used todetermine the prognostic potential of activated GTPase markers alone orin combination with downstream effector pathways.

Statistical Analysis.

Clinical results are analyzed statistically. Pearson correlation (r) areused to compare Rac1/Cdc42 staining intensity to that of other markers,and results are confirmed using Spearman correlation. For the remaininganalyses, staining intensity is dichotomized with high- oroverexpression defined as above the median of the product of intensitytimes the percentage of epithelial cells stained. For continuousdemographic data that are not normally distributed and for clinical andpathological data, respectively, nonparametric Wilcoxon and Fisher'sexact tests are used. The LIFETEST procedure is used to calculatesurvival curves, and differences in survival are compared using theLog-rank test. P values≦0.05 is considered statistically significant. Todiscern relationships between two target proteins (e.g. GTPase andspecific effectors) exhibiting positive staining in matched samples wewill stratify using the Nottingham Prognostic Index, which identifiesrisk using clinical and pathologic parameters groups, and then deriveCox proportional hazards and regression tree models. Training andvalidation sets are derived to test the ability to distinguish patientswith poor outcomes from those with good or moderate outcomes and tocalculate hazard ratios.

Results and Alternative Approaches

Rac1, Cdc42 and associated downstream effectors are predicted regulatorsof ovarian tumor cell growth and metastasis and we expect that inaddition to predicting poor overall survival, one or both of the GTPases(Rac1/Cdc42) along with PAK1 (or pPAK1), WAVE3, WASP or IQGAP1 areassociated with advanced stage and/or grade. If positive, we extend ourstudies to include analysis of matched primary tissue and metastaticlesions (US Biomax). Although our initial analysis of Rac1 and Cdc42 didnot reveal significant differences in expression between serous andendometrioid tumors (not shown), it is possible that histotypeassociations are identified with one or more markers. For example, IQGAPactivation leads to the disassembly of adherens junctions and nucleartranslocation of beta catenin [38]. Analyses of nuclear beta-cateninidentified a higher preponderance among endometriod as compared toserous carcinomas suggesting that activated beta-catenin mightdiscriminate between ovarian cancer subtypes [56]. Each of the proposedmarkers has been tested in human tumor tissues. We use only antibodiesthat have been validated for IHC and conduct studies to optimize eachmarker individually. Additional tissues are available through the HTR isadditional sample size is required to draw meaningful conclusions. PAK1expression levels and altered distribution between nucleus and cytoplasmare important in tamoxifen resistant breast tumors and glioblastomaswith poor prognosis [50; 57; 58]. Therefore, if PAK1 is a criticalfactor in ovarian cancer, expression is expected to be elevated and/ordistribution between cytoplasm and nucleus may be altered in ovariantumors as compared to normal tissues. As an alternative approach we lookfor altered PAK1 phosphorylation (correlated with poor outcome inglioblastoma) by western blot of primary tissue samples and taking intoconsideration best tissue procurement practices to preservephosphoprotein profiles [58; 59]. WAVE3, if crucial in ovarian cancer,is expected to be increasingly expressed in the cytoplasm of ovariantumor cells analogous to observations made for prostate cancer [51]. Assurrogates for WAVE3 activation, matrix metalloproteases (MMP) −1 and−9, but not −2 should be upregulated and active. Diffuse overexpressionof IQGAP1 is correlated with high-grade ovarian tumors, andoverexpression and increased membrane association are also linked togastric and hepatocellular carcinoma [60-62]. As a corollary to IQGAPactivation, we expect to find elevated nuclear beta-catenin. RT QPCR mayalso be used as an alternate or in parallel to protein expressionmeasures for marker validation. The outcomes from these studies willestablish essential relationships between GTPase activation andcorresponding downstream pathways that are related to ovarian tumor cellbehavior linked to metastatic dissemination. Importantly, these cellbehaviors are modulated by the R-enantiomers of naproxen and ketorolacand the Rac1 inhibitor NSC23766 (FIG. 4). The inventors thus identifythose pathways with greatest likelihood of response to GTPase targetedtherapeutics to be tested.

Determine the Mechanisms of Action of Small Molecule Inhibitors ofRac1/Cdc42 in Biochemical and Cell Based Assays. Rationale:

Chemical Library Screens are conducted to identify inhibitors andactivators of small GTPases [19]. Screening of the Prestwick chemicallibrary of out-of-patent drugs identified the R-enantiomer of naproxenas active against Rac1 and Cdc42, which was confirmed in cellularassays; GLISAs that measure GTPase activation status (FIG. 3A), andproliferation, adhesion and migration assays that are reflective ofpathways downstream of GTPase activation (FIG. 4). Using the Prestwickchemical library hit as a template, combined 2D- and 3D-based virtualscreening techniques [63] to identify additional NSAIDs (not in thelibrary) for testing was carried out. The nabumetone active metabolite,6-MNA, ranked first on this list, as it shares a common chemotype withR- and S-naproxen. Since 6-MNA lacked Rac1 and Cdc42 activity (FIG. 3Aand not shown), it was postulated that the rotational barrier around thecarboxyl-substituted chiral center may explain differences in GTPaseactivity. Therefore the inventors performed additional similarityqueries focused on a rotationally constrained carboxyl-substitutedchiral center, and identified R- and S-ketorolac (also not in library)as matching enantiomeric candidates. R-ketorolac inhibited ovarian tumorcell migration with an IC₅₀ of ˜10 μM (FIG. 4B).

Significant differences in the inhibitory activities of the naproxenseries against GTPases were quantified (R-naproxen>S-naproxen>6-MNA)(FIG. 4A). The differences are thought to be a consequence of bothrotational constraints around the chiral center imposed by the methylgroup and a requirement for the aryl rings to be accommodated in ahydrophobic pocket that has a pyrrolizine that restricts rotation of alinked benzoyl ring. This is exemplified by recent crystallographicstudies on naproxen binding to COX. S-naproxen displays optimal fit andinhibitory activity (50-70%) against COX enzymes based oncrystallographic and enzymatic evaluations [22]. In contrast, R-naproxendisplayed only 10-20% inhibition at >25 μM, which is explained based onthe limited structural modification permitted by the NSAID bindingpocket on COX enzymes [22]. In contrast to the COX enantiomericspecificity, virtual docking predicts R-naproxen, but not S-naproxenbinding to the GDP-bound pocket of Rac1 (FIG. 3B). The model forR-naproxen binding to Rac1 predicts stabilization of the GDP-boundGTPase and provides a testable, novel mechanism for the inhibitoryactivities of the R-enatiomers of naproxen and ketorolac.

General Approach:

In vitro and cell based assays are conducted to: 1) establish themechanism of naproxen and ketorolac inhibition using flow cytometrybased equilibrium and kinetic assays; 2) monitor potential disruption oftarget interaction with GTPase regulatory and effector molecules invitro; and 3) monitor drug treated cells for in vivo inhibition ofpathways that are directly downstream of Rac1 and Cdc42 activation.

Testing of Compounds

Compounds for analysis are summarized in Table 2. Additional compoundsare identified in FIG. 10. Each compound is tested in a log dose rangecorresponding to therapeutically relevant concentrations (average serumconcentrations at standard dosing). The range for cell based testing ofthe naproxen series (R-, S- and 6MNA) is 10-300 μM; ketorolacs (R- andS-) is 1-30 μM and NSC 23766 10-100 μM (positive control for Rac1inhibition). For in vitro assays the concentrations extend into thenanomolar range.

In Vitro Analyses of Nucleotide Binding to Determine Mechanism ofAction.

A bead-based flow cytometry assay for measuring nucleotide binding isused to test the predicted novel mechanism of drug interaction withpurified Rac1 and Cdc42 GTPases. Non-competitive and uncompetitivebinding mechanisms of NSAIDs and other small molecules to protein activesties are well described in the literature [64-68]. BODIPY(4,4-difluoro-4-bora-3a,4a-diaza-s-indacene)-labeled nucleotide (GDP orGTP) binding to GSH-bead immobilized GST-Rac or GST-Cdc42 isquantitatively measured by flow cytometry as previously described [19;69; 70]. Both equilibrium nucleotide binding in the presence or absenceof increasing doses of each test compound (Table 2) and kinetics ofnucleotide binding and dissociation with and without each compound ismeasured. The K_(d) for BODIPY-GTP/GDP of Rac1 and Cdc42 is ˜100 nM.Equilibrium competition experiments establish the EC₅₀ by testing serialdilutions of each compound across concentrations (0.03 nM-30 μM), whileholding the concentration of BODIPY GTP/GDP constant around the K_(d).Dissociation measurements under equilibrium conditions is performed byallowing BODIPY-nucleotide binding to saturation (90 min) and adding anexcess of unlabeled nucleotide or drug and monitoring the dissociationrate of the bound nucleotide in real time. Kinetic experiments measureBODIPY-GTP/GDP on rates over ˜250 s in the presence of compoundconcentrations well in excess of the EC₅₀. Off rates are determined byprebinding GTPase and BODIPY-GTP/GDP and adding compound in excess ofEC₅₀ concentration. DMSO and excess unlabeled GTP/GDP controls areincluded in all experiments. Data are normalized and analyzed usingPrism software as described in our published studies [19; 69; 70].

One of the predicted R-naproxen contact points is through a hydrogenbonding interaction with T17 of Rac1. Therefore, we test the impact ofthe classic dominant negative Rac T17N alone or in combination with D57Aand D57N mutants on R-naproxen binding tested using BODIPY-GDP and flowcytometry as described above. Depending on the outcomes of the mechanismof inhibition studies on ketorolac, virtual docking studies is conductedand relevant site-directed mutants is generated as illustrated forR-naproxen (FIG. 3B).

These studies use the flow cytometry based GTP binding assay used forthe initial HTS screen and characterization of other GTPase inhibitors[19; 69; 70], in establishing mechanism of drug interaction and insilico predictions of interactions to inform studies of protein: druginteraction [63; 76-84].

In Vitro Analyses of Drug Effects on Regulatory and Effector ProteinInteractions with GTPases.

The virtual docking studies predict a novel mechanism of R-naproxenaction (stabilizing inactive, GDP-bound state) relative to known Rac1inhibitors that bind to the switch regions and block guanine nucleotideexchange factor interactions or promote nucleotide release [17; 33; 89].GTPases have two switch regions that undergo significant conformationalchanges based on which nucleotide (GDP or GTP) is bound. Effectorbinding is restricted to the GTP-bound conformation thus we anticipatedisruption of this interaction. In vitro Pak effector binding assaysmeasure the inhibitory effects of drug binding on GTPase-effectorprotein interactions [90]. Purified His-Rac1 or His-Cdc42 is brieflypreincubated (<15 min) with each test compound (0-100 μM), +/−GDP orGTPγS addition. Recovery of GTPases in the active conformation is viaPAK coated ELISA plates (Cytoskeleton, Inc.) and luminescentGTPase-specific antibody based readout or via addition of GST-PAK-PBDimmobilized on GSH-beads followed by immunoblotting for bound GTPasefraction. Effects of naproxen and ketorolac on Tiara or Dbl GEF mediatedactivation of Rac1 and Cdc42, respectively, is measured using a kinetic,96-well plate based mant-GTP binding assay in routine use in the lab.Tiam GEF is measured in the presence of ascorbyl stearate [91]. Dbl andreagents for GEF assay are purchased from Cytoskeleton, Inc. NSC23766serves as a positive control for GEF inhibition.

Expected Results In Vitro Analysis:

The mechanism of inhibition of compounds according to the presentinvention are determined from flow cytometry experiments. In anuncompetitive inhibition mechanism the drug would bind only to thenucleotide-bound GTPase and thus would be expected to decrease theapparent K_(d) and decrease the B_(max). A noncompetitive inhibitionmechanism would allow both the nucleotide and drug to bindsimultaneously, but independently and thus without any change to theapparent K_(d). However, since the bound inhibitor would block enzymeactivity, B_(max) should decrease. A competitive mechanism is consideredunlikely, but would be associated with an increase in K_(d) and nochange in B_(max). Both Rac1 and Cdc42 bind to the PAK effector protein.Binding assays in the presence of GDP and GTP alone will provide minimumand maximum binding activities. Binding in the presence of increasingdoses of drugs will establish effects on regulatory and effector proteininteractions. The composite studies lend insight into mechanism ofaction and inform plans for crystallographic analyses. One-way ANOVA ofduplicates and repeated measures to 95% confidence intervals identifyactives.

Small Molecule Impact on Effector Activity in Cells.

GLISA is extensively used for measuring EOF-stimulated Rac and Cdc42activation in Swiss 3T3 cells in the presence and absence of testcompounds. Preliminary data demonstrate R-naproxen inhibits Rac1 andCdc42 GTPase activities in OvCA cell lines (429 and 433). However,direct GTPase inhibitory activity for the ketorolac series, althoughexpected based upon the initial results, remains to be established.GLISAs in are highly specific, quantitative, can be performed with smallsample sizes (<50 μg of cell lysate per assay, compared to 1-10 mgrequired for conventional PAK binding assay) and have excellentreproducibility. To assess the inhibitory effects of the small moleculesin Table 2, preincubate cells for 15-60 min (times that establishdetection of acute GTPase inhibition) with varying concentrations ofeach compound as described. NSC23766 serves as a positive control forRac1 inhibition. The optimal time for measuring maximal EGF-stimulatedRac and Cdc42 activation in Swiss 3T3 cells and OVCA429 cells is after 2min EOF treatment (FIG. 3A). Activated RhoA is also measured by GLISAfollowing lysophosphatidic acid stimulation as an additional specificitycontrol as needed. These analyses establish cellular IC50s (forR-naproxen and R-ketorolac relative to S-enantiomers and controlcompounds). Based on data, expectation of the enantiomer selectivity tobe R>S, the reverse of what has been established for the COX enzymes[22].

As a mechanistic corollary to the inhibitory properties of R-naproxenand R-ketorolac on cell behaviors (proliferation, adhesion andmigration, FIG. 4), it is of interest to determine which Rac1 and Cdc42regulated pathways are directly affected by GTPase inhibition (FIG. 1,Table 1). Preliminary data demonstrate that R-naproxen, but not 6MNAtreatment of OvCA cell lines inhibits Rac and Tiam membrane associationconsistent with the inhibition of Rac1 activity measured by GLISA (FIG.5A-C). In addition, the formation of Cdc42-dependent, actin-basedinvadopodia that are crucial for matrix degradation and invasion [92;93] is also significantly reduced by R-naproxen, but not by 6 MNA (FIG.5D-E).

Using a combination of immunofluorescence staining, western blotting tomonitor and quantify changes in localization, expression andphosphorylation of key downstream effectors of Rac1 and Cdc42+/−drugtreatment (6-24 h) are monitored. The markers to be studied are the sameas those that are monitored by IHC in tumor samples and allow directtesting of the impact of drug treatment on key pathways that aredownstream of GTPase activation to gauge how well they are targeted byGTPase inhibition. Molecules/pathways analyzed include: a) membranelocalization of GTPases (Rac1 and Cdc42) and regulatory proteins (Tiam1,Dbl GEFs) as measures of GTPase activation; b) IQGAP/beta cateninlocalization to adherens junction or nucleus as markers of stablecell-cell adhesion vs. EMT; c) PAK/pPAK as markers of pathwaysdownstream of Rac1 and Cdc42 that alter proliferation and migration; d)WAVE and WASP membrane association in conjunction with lamellopodia andfilopodia/invadopodia formation as measures of acute actin remodelingneeded for cell migration and invasion. SiRNA knockdown of Rac1 andCdc42 alone or in combination is used to verify that the drug responsesdepend on inhibition of Rac1 and Cdc42 function.

Expected Results and Alternatives:

The cell based assays identify which Rac1 and Cdc42 pathways are mostdirectly impacted by GTPase inhibition and together with IHC resultsobtained in Aim1 provide mechanistic information about the pathways thatare activated in ovarian cancer and can best be inhibited by smallmolecule GTPase inhibitors. In addition, the studies inform whichmeasures are most useful for tracking molecular analyses of xenografttissues as part of Aim 3. Requisite expertise in imaging and all thetools and assays needed for the study are routine as illustrated by datashown. Many commercial antibodies are available from different sources,thus, biochemical measures of phosphorylation or cell fractionation toestablish changes in localization may be used as alternates. Statisticsperformed as for in vitro analyses.

Testing the activity of select NSAIDs in preclinical cell and xenograftanimal models.

The ovarian tumor microenvironment contains small molecules and ligandsthat stimulate signaling pathways upstream of Rac1 and Cdc42 (FIG. 1)[35; 94] and adhesive events required for peritoneal implantation andinvasion also stimulate Rac1 and Cdc42 [95-102]. Therefore, it isbelieved that R-naproxen and R-ketorolac have demonstrable impact onbiological processes required for ovarian cancer metastatic success(FIG. 7). In support of this hypothesis, R-naproxen decreased the numberof implanted tumors in a xenograft animal model using athymic nude miceinjected with human ovarian tumor cells adapted to intraperitonealgrowth (SKOVip; obtained through a formal MTA from MD Anderson).Treatments were incorporated into transgenic dough (BioServe,Frenchtown, N.J.) and animals were dosed twice per day to achieveestablished therapeutic serum levels [73]. Animals were given placebodough or dough containing R-naproxen, S-naproxen or 6MNA. Aftersacrifice, animals fed R-naproxen exhibited a 4-fold reduction in tumornumber (FIG. 6) and a 36% decrease in total tumor burden (not shown),while the 6MNA and S-naproxen treated animals were similar to theplacebo controls. These data demonstrate that the enantiomer specificinhibitory properties of R-naproxen are preserved in vivo and that thisNSAID may reduce tumor growth and spread, a point that is further testedand extended to ketorolac.

Additional Research:

An organotypic model is used to study the impact of small molecules ondistinct components of ovarian tumor metastasis using cells derived frompatients (ex vivo assays) and established ovarian tumor xenograft models(in vive assays) (FIG. 6). Use of multiple ovarian cancer cell lines(429,433,Skov) and small molecule interventions defined in Table 2.Important questions regarding the biologic impact of small molecules ondistinct aspects of ovarian tumor metastasis (see also FIG. 7) areanswered:

1) R-Naproxen or Ketorolac Disrupts MCA Formation and Inhibits OvarianTumor Cell Proliferation and Survival in Suspension.

Metastatic cells are shed into the peritoneal cavity as single cells andspheroids or multicellular aggregates (MCAs) [103; 104] and because themajority of conventional chemotherapies are ineffective in preventingMCA growth and dissemination, they are considered a likely source ofrecurrent disease [105]. Disrupting MCA behavior is believed torepresent an important therapeutic strategy.

2) The Small Molecules Interfere with Ovarian Tumor Cell Attachment andInvasive Anchoring to the Mesothelium Required for Metastatic Growth.

Aggregation and peritoneal attachment of multicellular aggregates (MCAs)is mediated via both β1-integrin and E-cadherin-dependent mechanisms[104; 106-112]. Rho-family GTPases are implicated in the establishment,maintenance and stability of cell:cell contacts and cadherin or CD44engagement stimulates Rac1 and Cdc42 activity [95-102]. This illustratesthe importance of testing small molecule inhibitors of Rac1/Cdc42 oncell:cell interactions that are meaningful for ovarian cancermetastasis.

Ex vivo Studies:

The impact of novel small molecules on patient-derived tumor cells exvivo is tested. Under an approved protocol, collection of tissues andascites from patients with primary ovarian cancer is performed. Thetissue is de-identified and the presence and type of tumor is verifiedhistologically by a pathologist. Fresh tissue offers several distinctadvantages to cell lines, namely the ability to 1) investigatedose-dependent response of primary ovarian tumor cells as a precursor tohuman translational studies, 2) directly relate target expression andactivity with cell response to test compounds, and 3) establish whetherthe responses obtained in homogeneous established cell lines (FIG. 4)are replicated in the more relevant, heterogenous cell populationsisolated from patients. OVCA 429 and SKOV3ip cells are included inparallel as these cells are used in xenograft models [113-117] forcomparison between in vitro response and the in vivo correlates.Organotypic cultures and 3D collagen gels (3 DCI) are used to modeladhesive events that occur during intraperitoneal dissemination in vivo.

Functional Studies:

Ovarian ascites tumor cells are routinely maintained in short termculture with samples from ˜35-75% of patients able to be established forex vivo analysis [118]. Similarly, cultures can be developed from mincedprimary tumor tissue [119]. The epithelial nature of the cells isconfirmed by EpCAM and HE4 antibody immunostaining. Only primary linesconfirmed as greater than 80%6 epithelial tumor cells after enrichmentare selected for analysis. Cell response −/+ the test compounds iscross-referenced to target expression and/or activity as in Aims 1-2.Standard experimental approaches which are used are demonstrated in theinventors' publications and preliminary findings [120-125]. Briefly,proliferation is measured by MTS assay, BrdU incorporation and cellcounts; apoptosis by flow cytometry; migration and invasion by cellmovement through a modified Boyden chamber without or with an artificialbasement membrane, respectively; and cell aggregation/formation of MCAformation is evaluated in a hanging drop assay and MCA size and shape isanalyzed by morphometric analysis [123]. Aggregate stability isestimated by subjecting MCAs to shear stress (trituration) followed bymorphometric analysis [126]. MCAs is formalin fixed, paraffin embedded,sectioned and stained for active caspase-3 and -7 (BD Biosciences andCell Signaling Technology, respectively) and cleaved PARP (CellSignaling) or ApopTag reagents (Millipore). An apoptotic index isdetermined by quantifying the number of apoptotic cells as a fraction ofthe total cell number [127]. Control sections are stained withhematoxylin and eosin and Ki67 to quantify cell proliferation.

To more closely approximate adhesive events that occur duringintraperitoneal dissemination in vivo uses organotypic cultures [128].Organotypic cultures and 3DCI gels are generated as previously described[35; 128-131]. Briefly, type I collagen gels with embedded fibroblastsare overlaid with a confluent layer of mesothelial cells (LP9) in 24well plates or transwells containing a porous filter. Alternatively,3DCI gels are evaluated in the absence of mesothelial cells to mimicmesothelial cell retraction and exposure of the underlying ECM. Adhesionand invasion of cells labeled using membrane permeable fluorescent dyes(CMFDA, Invitrogen) are enumerated using fluorescent microscopy [128;131]. Expansive growth in 3D collagen is measured after cell growth for6-10 d using morphometric analysis of proliferative aggregate dimensionsand quantitation of the number and size invasive projections, asdescribed previously [123].

Statistical Analysis.

Measurements are summarized using means, standard deviations (SD) and95% confidence intervals (CIs). Categorical variables are summarized aspercentages with 95% CIs. Box plots are used to informally comparegroups and to check the normality assumption needed for parametricanalyses. All tests are conducted at the 5% level. Multiple comparisons(i.e. Tukey's test) are used to quantify group differences whendifferences among groups are statistically significant. Analysis ofvariance (ANOVA) is used to compare means among treatment and controlconditions when the basic unit of analysis is a measurement or anaverage. Analogous methods is used for counts (i.e. Poisson regression)and proportions (i.e. logistic regression for apoptotic index).

In Vivo Studies

GFP-expressing OVCA 429 and SKOV3ip cells are used for theintraperitoneal (IP) xenograft studies (FIG. 6). The IP model will allowus to focus on adhesion and invasion in addition to tumor growthcharacteristics. Animals injected with SKOV3ip cells typically display˜20-30 metastatic implants at 20 days post implantation and animalssuccumb to disease at ˜40 days with 80-100 overt peritoneal metastases[115]. Mice inoculated with OVCA 429 cells display a phenotype of large(>1.5 cm³) solid tumors that adhere loosely to fat in the pelvic region,intestine, and/or omentum in addition to multiple smaller tumors (<0.25cm3) accumulating in the same three areas, with ascitic fluidaccumulation [117]. Two treatment procedures are followed. 1) Introducetumor cells and allow 7 days for the cells to engraft before treatmentas a model of small molecule impact on residual disease. 2) Mice receivethe intervention for two days before tumor cell inoculation toapproximate the impact of small molecules on recurrent disease includingtumor growth, metastatic implantation and invasive peritoneal lesions.The dose of R-naproxen, S-naproxen and 6-MNA is 20 mg/kg (oral) toapproximate prescription dose levels in humans and these are commondoses for naproxen treatment in mice [132-136]. Treatment of mice with10 mg/kg S-naproxen led to plasma concentrations of ˜94 μM within 3 h[137]. Ketorolac is dosed orally at 1 mg/kg to achieve serum levelscomparable to human therapeutic doses [23; 75]. Based on publishedstudies of in vivo NSC23766 administration, subcutaneous infusion via anosmotic minipump provides effective dosing [138; 139]. A dose of 10mg/kg/day reduced Rac1 activity in the target tissue (kidney) withoutapparent evidence of organ toxicity in mice following 6 weeks ofcontinuous dosing [139]. Saline administration via osmotic minipumpserves as the control for NSC23766. These studies represent an extensiveevaluation of drug action on growth and metastatic success, which hasnot been evaluated for naproxen or ketorolac or known Rac inhibitors inovarian or other cancers.

Tumor Analysis

Tumor growth is estimated three times per week by weight and abdominalcircumference in addition to GFP imaging using an MS Lumina II. Atsacrifice ovarian tumor burden for each treatment condition is obtainedby dissecting solid tumors from the peritoneal cavity, including tumorcell volume recovered from ascites and peritoneal washings [117].Evaluate in some detail of tumor burden including ascites tumor, as wellas metastatic events such as peritoneal adhesion and anchoring.Metastatic disease is to be scored based on pattern of spread, number,and size of secondary foci. Specifically, the number of lesions at eachorgan site (bowel, mesentery, pancreas, liver, diaphragm) is quantifiedand categorized as small (<1 mm), medium (1-2 mm) or large (>2 mm). Incollaboration with Pathologist Lomo, tumor-containing abdominal tissuesis fixed in formalin, paraffin embedded, sectioned and stained with H&Eto enable scoring of the extent of invasion [117] on a 1-3 scale, where1=non-invasive tumors with a smooth tumor-mesothelial interface,2=moderately invasive with some infiltration of underlying tissue and3-highly invasive, with significant submesothelial growth and evidenceof desmoplastic host response [131].

Tumor Tissue Analysis

Excised tumors are evaluated for target enzyme inhibition, effectoranalysis, proliferation, apoptosis, and angiogenesis. Followingsacrifice, tumor tissue is collected and then fixed in 10%phosphate-buffered formalin solution for immunohistology or snap frozenin liquid nitrogen then stored at −80° C. until analyzed. Tumor massesfrom and ascites cells is analyzed for GTPase activity by GLISA assay(Rac1, Cdc42), COX activity, proliferation index, apoptotic index, andmean vessel density. The (LISA assays have been used successfully inisolated tissues (Cytoskeleton, Inc) and would provide a direct measureof effective target inhibition. Alternatively, if greater tissue sampleis required, Rac1 and Cdc42 activity is measured by Pak1 pull downassays from tumor lysates. COX activity is measured by ELISA.Proliferation and apoptotic indices based on tissue staining for Ki67and TUNEL staining, respectively. The proliferation index is defined aspercent cells positive for Ki67 in five randomly selected high powerfields per tumor. Apoptotic index is determined by the number ofapoptotic tumor cells in five randomly selected high-power fieldsexclusive of necrotic areas. Because COXs and Rho-family GTPases areinvolved in angiogenesis [140-148], we will measure mean vessel density(MVD) by counting CD31-positive vessels as described previously [149].Analyses of the expression and localization of downstream effectors(PAK1, WAVB3 and IQGAP1) as a function of drug treatment

Statistical Analysis.

For animal studies, a 5% ANOVA test of no differences in means based ona sample of 8 per group has 82% (81%) power to detect a moderate effectsize of 0.60 (0.63), where the effect size is given by the SD of thegroup means divided by the within group SD. Kaplan-Meier survival curvesand log-rank tests is used to compare time to events. Repeated measuresANOVA are used to compare longitudinally measured in vivo growthparameters. Parametric or non-parametric ANOVA is used to compare tumorgrowth, immunochemistry and tissue measurements across groups at eachtime point. The mean number of lesions is compared across groups usingANOVA, treating the responses as Poisson counts. The extent of invasionand size of lesions are categorical responses and is compared acrossgroups using chi-squared tests of homogeneity and logistic regression,respectively. Multiple lesions per site is handled using generalizedestimating equations to give valid statistical tests that account forwithin-individual clustering. Tumor tissue measurements are summarizedtreated as for in vitro cell measurements.

Results and Alternative Approaches

The studies focus on the impact of identified small molecules ondistinct mechanisms of metastasis in ovarian cancer (FIG. 7) and obtaindetailed information adhesive events (cell:cell for MCAs orcell:mesothelium ex vivo and in vivo), invasion, growth and survival.Parallel experiments conducted in monolayer culture assessproliferation, apoptosis, migration, in vitro invasion andquantification of E-cadherin surface expression and endocytosis usingtechniques and approaches that are well documented in publications[121-125; 150; 151] to complement the findings obtained in 3D andorganotypic models. Based on our preliminary findings, we predict thatthe R-enantiomers and NSC23776 will decrease MCA formation, cellmigration and cell invasion because actin nucleation and polymerizationare proximal targets for Rac1/Cdc42 through activation of effectormolecules such as IQGAP & PAKs. However, MMPs are additionally requiredfor invasion and COX activity is implicated in MMP expression [151] so6MNA and the S-enantiomers may partially block invasive anchoring. Wepredict that the impact of small GTPase inhibition by NSC23766,R-naproxen or R-ketorolac will be most evident in the IP metastaticmodels where Rac1 and Cdc42 signaling play key roles in adhesive andactin-cytoskeleton-mediated events crucial to metastatic success (suchas implantation and invasive lesions. The information on effectiveconcentrations guides dose parameters for the future transition to Phase1/2 clinical trials. If direct measurements of Rac1/Cdc42 activity inconjunction with effector activation reveal poor inhibition, we willadjust doses accordingly. While not all patient-based tumor cells willestablish in short term culture or xenograft models, but with 60cases/year the numbers are sufficient to validate the impact of thenovel drugs on tumor-relevant endpoints. Promising results ex vivo andwith tumor cell lines in vivo, will extend the studies to include IPxenograft studies using primary tumor cells from patients.

One concern is that S-ketorolac may cause gastrointestinal lesions inthe course of the in vivo studies since these experiments will exceedthe length of time recommended for human use of ketorolac. The GItoxicity in mice is dose dependent and low, but detectable at theproposed dose [23; 75]. Necropsy and histopathology are performed on anyanimals that succumb before conclusion of the tumor studies usingservices provided through the Cancer Center Animal Research Core. We donot anticipate problems with R-ketorolac because there is littleinterconversion between the R- and S-forms in rodents, and nonedetectable in humans [26]. Similarly, S-naproxen is not significantlysubject to epimerization [29].

Because R-naproxen inhibits Rac1 and Cdc42, these studies will notclarify whether targeted inhibition of Rac1 or Cdc42 is moreadvantageous for antitumor activity in ovarian cancer. If antitumoractivity is verified, follow up is with targeted knockdown of Rac1 orCdc42 using Tet-inducible shRNA (ClonTech) or lentiviral approaches tocompare the impact of targeting the GTPases singly or in combination. Werecognize that as is true for all small molecules, the test compoundsmay have additional, as yet unidentified targets that could contributeto biologic response. By using positive controls for small GTPases(NSC23766, established Rac1 inhibitor), controls for response due to COXinhibition and incorporation of Rac1 or Cdc42 knockdown strategies weare able to address this possibility. In addition, a future goal is totest relevant small molecules in the TgMISIIR TAg mouse model of ovariancancer to highlight potential impact on early disease.

Summary

The experimental studies described herein represent the first efforts toinvestigate the potential of Rac1 and Cdc42 as therapeutic targets inmetastatic ovarian cancer. The compounds identified, among othersdescribed, are FDA approved as the racemic mixture. Therefore, proof ofconcept that therapeutic targeting of Rac1 and/or Cdc42 confersanti-tumor benefit for ovarian cancer in vivo offer the potential forrapid clinical translation. An observational report notes thatpresurgical administration of R,S-ketorolac reduced cancer recurrencerates in breast cancer patients [152]. Thus, suggesting even short-termadministration of racemic mixtures are of benefit. Establishing theR-enantiomers as drugs with novel targets combined with the possibilityof obtaining R-enantiomer pure compounds for clinical studies throughthe NCI's Experimental Therapeutics (NExT) program will open newopportunities for drug development in a disease with limited options.

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1. (canceled)
 2. A method of treating ovarian cancer in a human patientin need thereof, comprising the steps of: (a) measuring the expressionor activity of Rac1 or Cdc42 in a sample of ovarian cancer tissueobtained from the patient; and (b) if Rac1 or Cdc42 in the sample isoverexpressed or activated compared to the expression or activity innon-cancerous tissue, administering to said patient an anti-cancereffective amount of a composition comprising ketorolac, or apharmaceutically acceptable salt thereof in combination with apharmaceutically acceptable carrier, additive or excipient, wherein theketorolac comprises an effective amount of R-ketorolac.
 3. The method ofclaim 2 wherein the ketorolac is racemic.
 4. The method of claim 2wherein the ketorolac is R-ketorolac.
 5. The method of claim 4 whereinthe R-ketorolac is enantiomerically pure.
 6. The method of claim 2wherein the ovarian cancer of the patient has increased expression oractivity of Rac1.
 7. The method of claim 6 wherein the expression ofRac1 by performing immunohistochemical analysis on the tissue.
 8. Themethod of claim 6 wherein the expression of Rac1 is measured bymeasuring Rac1 mRNA using quantitative PCR analysis.
 9. The method ofclaim 6 wherein the activity of Rac1 is measured by performing a GLISAactivity assay for Rac1 GTPase activity.